Vaccine compositions comprising a saponin adjuvant

ABSTRACT

The present invention provides a human dose of an immunogenic composition comprising an antigen or antigenic preparation, in combination with an adjuvant which adjuvant comprises an immunologically active saponin fraction derived from the bark of  Quillaja Saponaria  Molina presented in the form of a liposome and a lipopolysaccharide wherein said saponin fraction and said lipopolysaccharide are both present in said human dose at a level of below 30 μg. The present invention further provides an adjuvant composition in a human dose suitable volume comprising between 1 and 30 μg of a lipopolysaccharide and between 1 and 30 μg of an immunologically active saponin fraction presented in the form of a liposome.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 12/096,838filed Jun. 10, 2008, now abandoned, which was filed pursuant to 35 USC371 as a United States National Phase Application of InternationalPatent Application Serial No. PCT/GB2006/004634 filed on Dec. 12, 2006,which claims the priority of 0525321.6 filed on Dec. 13, 2005, 0609902.2filed on May 18, 2006, 0620336.8 filed on Oct. 12, 2006, 0620337.6 filedon Oct. 12, 2006 in the United Kingdom which are incorporated herein intheir entirety.

STATEMENT REGARDING SEQUENCE LISTING

Applicants respectfully request that the Sequence Listing provided inelectronic form (as.txt file) in lieu of a paper copy, be incorporatedby reference into the specification. The name of the text filecontaining the Sequence Listing is VB61750C1_Sequence_Listing.txt. It ishereby stated that the content of the Sequence Listing does not includenew matter.

FIELD OF THE INVENTION

The present invention relates to improved vaccine compositions, methodsfor making them, and their use in medicine. In particular the inventionrelates to adjuvanted vaccine compositions wherein the adjuvant is aliposomal formulation, comprising a saponin and a lipopolysaccharide.The present invention further relates to influenza vaccine formulationsand vaccination regimes for immunizing against influenza disease.

BACKGROUND OF THE INVENTION

New compositions or vaccines with an improved immunogenicity are alwaysneeded. As one strategy, adjuvants have been used to try and improve theimmune response raised to any given antigen.

Lipopolysaccharides (LPS) are the major surface molecule of, and occurexclusively in, the external leaflet of the outer membrane ofgram-negative bacteria. LPS impede destruction of bacteria by serumcomplements and phagocytic cells, and are involved in adherence forcolonisation. LPS are a group of structurally related complex moleculesof approximately 10,000 Daltons in size and consist of three covalentlylinked regions:

-   -   (i) an O-specific polysaccharide chain (O-antigen) at the outer        region    -   (ii) a core oligosaccharide central region    -   (iii) lipid A—the innermost region which serves as the        hydrophobic anchor, it comprises glucosamine disaccharide units        which carry long chain fatty acids.

The biological activities of LPS, such as lethal toxicity, pyrogenicityand adjuvanticity, have been shown to be related to the lipid A moiety.In contrast, immunogenicity is associated with the O-specificpolysaccharide component (O-antigen). Both LPS and lipid A have longbeen known for their strong adjuvant effects, but the high toxicity ofthese molecules has precluded their use in vaccine formulations.Significant effort has therefore been made towards reducing the toxicityof LPS or lipid A while maintaining their adjuvanticity.

The Salmonella minnesota mutant R595 was isolated in 1966 from a cultureof the parent (smooth) strain (Luderitz et al. 1966 Ann. N. Y. Acad.Sci. 133:349-374). The colonies selected were screened for theirsusceptibility to lysis by a panel of phages, and only those coloniesthat displayed a narrow range of sensitivity (susceptible to one or twophages only) were selected for further study. This effort led to theisolation of a deep rough mutant strain which is defective in LPSbiosynthesis and referred to as S. minnesota R595.

In comparison to other LPS, those produced by the mutant S. minnesotaR595 have a relatively simple structure.

-   -   (i) they contain no O-specific region—a characteristic which is        responsible for the shift from the wild type smooth phenotype to        the mutant rough phenotype and results in a loss of virulence    -   (ii) the core region is very short—this characteristic increases        the strain susceptibility to a variety of chemicals    -   (iii) the lipid A moiety is highly acylated with up to 7 fatty        acids.

4′-monophosporyl lipid A (MPL), which may be obtained by the acidhydrolysis of LPS extracted from a deep rough mutant strain ofgram-negative bacteria, retains the adjuvant properties of LPS whiledemonstrating a toxicity which is reduced by a factor of more than 1000(as measured by lethal dose in chick embryo eggs) (Johnson et al. 1987Rev. Infect. Dis. 9 Suppl:S512-S516). LPS is typically refluxed inmineral acid solutions of moderate strength (e.g. 0.1 M HCl) for aperiod of approximately 30 minutes. This process results indephosphorylation at the 1 position, and decarbohydration at the 6′position, yielding MPL.

3-O-deacylated monophosphoryl lipid A (3D-MPL), which may be obtained bymild alkaline hydrolysis of MPL, has a further reduced toxicity whileagain maintaining adjuvanticity, see U.S. Pat. No. 4,912,094 (RibiImmunochemicals). Alkaline hydrolysis is typically performed in organicsolvent, such as a mixture of chloroform/methanol, by saturation with anaqueous solution of weak base, such as 0.5 M sodium carbonate at pH10.5.

Further information on the preparation of 3D-MPL is available in, forexample, U.S. Pat. No. 4,912,094 and WO02/078637 (Corixa Corporation).

Quillaja saponins are a mixture of triterpene glycosides extracted fromthe bark of the tree Quillaja saponaria. Crude saponins have beenextensively employed as veterinary adjuvants. Quil-A is a partiallypurified aqueous extract of the Quillaja saponin material. QS21 is aHplc purified non toxic fraction of Quil A and its method of itsproduction is disclosed (as QA21) in U.S. Pat. No. 5,057,540.

By way of example, influenza vaccines and vaccines against humanpapilloma virus (HPV) have been developed with adjuvants.

Influenza viruses are one of the most ubiquitous viruses present in theworld, affecting both humans and livestock. Influenza results in aneconomic burden, morbidity and even mortality, which are significant.

The influenza virus is an RNA enveloped virus with a particle size ofabout 125 nm in diameter. It consists basically of an internalnucleocapsid or core of ribonucleic acid (RNA) associated withnucleoprotein, surrounded by a viral envelope with a lipid bilayerstructure and external glycoproteins. The inner layer of the viralenvelope is composed predominantly of matrix proteins and the outerlayer mostly of host-derived lipid material. Influenza virus comprisestwo surface antigens, glycoproteins neuraminidase (NA) andhaemagglutinin (HA), which appear as spikes, 10 to 12 nm long, at thesurface of the particles. It is these surface proteins, particularly thehaemagglutinin that determine the antigenic specificity of the influenzasubtypes.

These surface antigens progressively, sometimes rapidly, undergo somechanges leading to the antigenic variations in influenza. Theseantigenic changes, called ‘drifts’ and ‘shifts’ are unpredictable andmay have a dramatic impact from an immunological point of view as theyeventually lead to the emergence of new influenza strains that enablethe virus to escape the immune system causing the well known, almostannual, epidemics.

The influenza virus strains to be incorporated into influenza vaccineeach season are determined by the World Health Organisation incollaboration with national health authorities and vaccinemanufacturers.

HA is the most important antigen in defining the serological specificityof the different influenza strains. This 75-80 kD protein containsnumerous antigenic determinants, several of which are in regions thatundergo sequence changes in different strains (strain-specificdeterminants) and others in regions which are common to many HAmolecules (common to determinants).

Influenza viruses cause epidemics almost every winter, with infectionrates for type A or B virus as high as 40% over a six-week period.Influenza infection results in various disease states, from asub-clinical infection through mild upper respiratory infection to asevere viral pneumonia. Typical influenza epidemics cause increases inincidence of pneumonia and lower respiratory disease as witnessed byincreased rates of hospitalization or mortality. The severity of thedisease is primarily determined by the age of the host, his immunestatus and the site of infection.

Elderly people, 65 years old and over, are especially vulnerable,accounting for 80-90% of all influenza-related deaths in developedcountries. Individuals with underlying chronic diseases are also mostlikely to experience such complications. Young infants also may suffersevere disease. These groups in particular therefore need to beprotected. Besides these ‘at risk’-groups, the health authorities arealso recommending to vaccinate healthy adults who are in contact withelderly persons.

Vaccination plays a critical role in controlling annual influenzaepidemics. Currently available influenza vaccines are either inactivatedor live attenuated influenza vaccine. Inactivated flu vaccines arecomposed of three possible forms of antigen preparation: inactivatedwhole virus, sub-virions where purified virus particles are disruptedwith detergents or other reagents to solubilise the lipid envelope(so-called “split” vaccine) or purified HA and NA (subunit vaccine).These inactivated vaccines are given intramuscularly (i.m.) orintranasaly (i.n.).

Influenza vaccines, of all kinds, are usually trivalent vaccines. Theygenerally contain antigens derived from two influenza A virus strainsand one influenza B strain. A standard 0.5 ml injectable dose in mostcases contains 15 μg of haemagglutinin antigen component from eachstrain, as measured by single radial immunodiffusion (SRD) (J. M. Woodet al.: An improved single radial immunodiffusion technique for theassay of influenza haemagglutinin antigen: adaptation for potencydetermination of inactivated whole virus and subunit vaccines. J. Biol.Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborativestudy of single radial diffusion and immunoelectrophoresis techniquesfor the assay of haemagglutinin antigen of influenza virus. J. Biol.Stand. 9 (1981) 317-330).

Influenza vaccines currently available are considered safe in all agegroups (De Donato et al. 1999, Vaccine, 17, 3094-3101). However, thereis little evidence that current influenza vaccines work in smallchildren under two years of age. Furthermore, reported rates of vaccineefficacy for prevention of typical confirmed influenza illness are23-72% for the elderly, which are significantly lower than the 60-90%efficacy rates reported for younger adults (Govaert, 1994, J. Am. Med.Assoc., 21, 166-1665; Gross, 1995, Ann Intern. Med. 123, 523-527). Theeffectiveness of an influenza vaccine has been shown to correlate withserum titres of hemagglutination inhibition (HI) antibodies to the viralstrain, and several studies have found that older adults exhibit lowerHI titres after influenza immunisation than do younger adults (Murasko,2002, Experimental gerontology, 37, 427-439).

New vaccines with an improved immunogenicity are therefore still needed.Formulation of vaccine antigen with potent adjuvants is a possibleapproach for enhancing immune responses to subvirion antigens.

A sub-unit influenza vaccine adjuvanted with the adjuvant MF59, in theform of an oil-in-water emulsion is commercially available, and hasdemonstrated its ability to induce a higher antibody titer than thatobtained with the non-adjuvanted sub-unit vaccine (De Donato et al.1999, Vaccine, 17, 3094-3101). However, in a later publication, the samevaccine has not demonstrated its improved profile compared to anon-adjuvanted split vaccine (Puig-Barbera et al., 2004, Vaccine 23,283-289).

By way of background, during inter-pandemic periods, influenza virusescirculate that are related to those from the preceding epidemic. Theviruses spread among people with varying levels of immunity frominfections earlier in life. Such circulation, over a period of usually2-3 years, promotes the selection of new strains that have changedenough to cause an epidemic again among the general population; thisprocess is termed ‘antigenic drift’. ‘Drift variants’ may have differentimpacts in different communities, regions, countries or continents inany one year, although over several years their overall impact is oftensimilar.

In other words, an influenza pandemics occurs when a new influenza virusappears against which the human population has no immunity. Typicalinfluenza epidemics cause increases in incidence of pneumonia and lowerrespiratory disease as witnessed by increased rates of hospitalisationor mortality. The elderly or those with underlying chronic diseases aremost likely to experience such complications, but young infants also maysuffer severe disease.

At unpredictable intervals, novel influenza viruses emerge with a keysurface antigen, the haemagglutinin, of a totally different subtype fromstrains circulating the season before. Here, the resulting antigens canvary from 20% to 50% from the corresponding protein of strains that werepreviously circulating in humans. This can result in virus escaping‘herd immunity’ and establishing pandemics. This phenomenon is called‘antigenic shift’. It is thought that at least in the past pandemicshave occurred when an influenza virus from a different species, such asan avian or a porcine influenza virus, has crossed the species barrier.If such viruses have the potential to spread from person to person, theymay spread worldwide within a few months to a year, resulting in apandemic. For example, in 1957 (Asian Flu pandemic), viruses of the H2N2subtype replaced H1N1 viruses that had been circulating in the humanpopulation since at least 1918 when the virus was first isolated. The H2HA and N2 NA underwent antigenic drift between 1957 and 1968 until theHA was replaced in 1968 (Hong-Kong Flu pandemic) by the emergence of theH3N2 influenza subtype, after which the N2 NA continued to drift alongwith the H3 HA (Nakajima et al., 1991, Epidemiol. Infect. 106, 383-395).

The features of an influenza virus strain that give it the potential tocause a pandemic outbreak are: it contains a new haemagglutinin comparedto the haemagglutinin in the currently circulating strains, which may ornot be accompanied by a change in neuraminidase subtype; it is capableof being transmitted horizontally in the human population; and it ispathogenic for humans. A new haemagglutinin may be one which has notbeen evident in the human population for an extended period of time,probably a number of decades, such as H2. Or it may be a haemagglutininthat has not been circulating in the human population before, forexample H5, H9, H7 or H6 which are found in birds. In either case themajority, or at least a large proportion of, or even the entirepopulation has not previously encountered the antigen and isimmunologically naïve to it.

Papillomaviruses are small DNA tumour viruses, which are highly speciesspecific. So far, over 100 individual human papillomavirus (HPV)genotypes have been described. HPVs are generally specific either forthe skin (e.g. HPV-1 and -2) or mucosal surfaces (e.g. HPV-6 and -11)and usually cause benign tumours (warts) that persist for several monthsor years. Such benign tumours may be distressing for the individualsconcerned but tend not to be life threatening, with a few exceptions.

Some HPVs are also associated with cancers. The strongest positiveassociation between an HPV and human cancer is that which exists betweenHPV-16 and HPV-18 and cervical carcinoma. Cervical cancer is the mostcommon malignancy in developing countries, with about 500,000 new casesoccurring in the world each year. It is now technically feasible toactively combat primary HPV-16 infections, and even establishedHPV-16-containing cancers, using vaccines. For a review on the prospectsfor prophylactic and therapeutic vaccination against HPV-16 see CasonJ., Clin. Immunother. 1994; 1(4) 293-306 and Hagenesee M. E., Infectionsin Medicine 1997 14(7) 555-556, 559-564.

Although minor variations do occur, all HPV genomes described have atleast eight early genes, E1 to E8 and two late genes L1 and L2. Inaddition, an upstream regulatory region harbors the regulatory sequenceswhich appear to control most transcriptional events of the HPV genome.

HPV L1 based vaccines are disclosed in WO94/00152, WO94/20137,WO93/02184 and WO94/05792. Such a vaccine can comprise the L1 antigen asa monomer, a capsomer or a virus like particle. Methods for thepreparation of VLPs are well known in the art, and include VLPdisassembly-reassembly approaches to provide enhanced homogeneity, forexample as described in WO9913056 and U.S. Pat. No. 6,245,568. Suchparticles may additionally comprise L2 proteins. L2 based vaccines aredescribed, for example, in WO93/00436. Other HPV vaccine approaches arebased on the early proteins, such as E7 or fusion proteins such asL2-E7.

There is still a need for improved vaccines, especially in the case ofinfluenza and in particular influenza pandemics and for the elderlypopulation, or in the case of HPV vaccines.

Adjuvants containing combinations of lipopolysaccharide and Quillajasaponins have been disclosed previously, for example in EP0671948. Thispatent demonstrated a strong synergy when a lipopolysaccharide (3D-MPL)was combined with a Quillaja saponin (QS21). It has now been found thatgood adjuvant properties may be achieved with combinations oflipopolysaccharide and quillaja saponin as immunostimulants in anadjuvant composition even when the immunostimulants are present at lowamounts in a human dose.

SUMMARY OF THE INVENTION

In first aspect of the present invention, there is provided animmunogenic composition comprising an antigen or antigenic preparationthereof, in combination with an adjuvant composition comprising animmunologically active saponin fraction derived from the bark ofQuillaja Saponaria Molina presented in the form of a liposome and alipopolysaccharide.

In a second aspect of the present invention, there is provided animmunogenic composition comprising an influenza virus or antigenicpreparation thereof, in combination with a saponin adjuvant presented inthe form of a liposome. In a specific embodiment of this aspect, theimmunogenic composition further comprises a Lipid A derivative, such as3D-MPL.

Suitably the saponin adjuvant in the form of a liposome according to theinvention comprises an active fraction of the saponin derived from thebark of Quillaja Saponaria Molina, such as QS21, and a sterol, such ascholesterol, in a ratio saponin:sterol from 1:1 to 1:100 w/w.

In particular, said immunogenic composition comprises an antigen with aCD4 T cell epitope. Alternatively, said immunogenic compositioncomprises an antigen with a B cell epitope.

The invention also relates to the use of an influenza virus or antigenicpreparation thereof, and an adjuvant comprising an immunologicallyactive saponin fraction derived from the bark of Quillaja SaponariaMolina presented in the form of a liposome and a lipopolysaccharide inthe manufacture of an immunogenic composition for the prevention ofinfluenza virus infection and/or disease.

The invention also relates to the use of a human papilloma virus antigenor antigens or antigenic preparation thereof, and an adjuvant comprisingan immunologically active saponin fraction derived from the bark ofQuillaja Saponaria Molina presented in the form of a liposome and alipopolysaccharide in the manufacture of an immunogenic composition forthe prevention of human papilloma virus infection and/or disease.

The invention also relates to the use of a Cytomegalovirus antigen orantigens or antigenic preparation thereof, and an adjuvant comprising animmunologically active saponin fraction derived from the bark ofQuillaja Saponaria Molina presented in the form of a liposome and alipopolysaccharide in the manufacture of an immunogenic composition forthe prevention of Cytomegalovirus infection and/or disease.

The invention also relates to the use of a Streptococcus pneumoanieantigen or antigens or antigenic preparation thereof, and an adjuvantcomprising an immunologically active saponin fraction derived from thebark of Quillaja Saponaria Molina presented in the form of a liposomeand a lipopolysaccharide defined in the manufacture of an immunogeniccomposition for the prevention of Streptococcus pneumonaie infectionand/or disease.

The invention also relates to the use of a Plasmodium falciparum antigenor antigens or antigenic preparation thereof, and an adjuvant comprisingan immunologically active saponin fraction derived from the bark ofQuillaja Saponaria Molina presented in the form of a liposome and alipopolysaccharide in the manufacture of an immunogenic composition forthe prevention of Plasmodium falciparum infection and/or malarialdisease.

The invention also relates to the use of a Varicella Zoster virusantigen or antigens or antigenic preparation thereof, and an adjuvantcomprising an immunologically active saponin fraction derived from thebark of Quillaja Saponaria Molina presented in the form of a liposomeand a lipopolysaccharide in the manufacture of an immunogeniccomposition for the prevention of Varicella Zoster virus infectionand/or disease.

In another aspect there is provided the use of (a) an antigen orantigenic preparation thereof, and (b) an adjuvant as hereinabovedefined in the manufacture of an immunogenic composition for inducing,in a human, at least one, or at least two, or all of the following: (i)an improved CD4 T-cell immune response against said antigen or antigenicpreparation thereof, (ii) an improved humoral immune response againstsaid antigen or antigenic preparation thereof, (iii) an improvedB-memory cell response against said antigen or antigenic preparationthereof.

In particular said antigen is an influenza virus, HPV, Cytomegalovirus(CMV), Varicella zoster virus (VZV), Streptococcus pneumoniae or malariaantigen or antigenic preparation thereof, and said human is animmuno-compromised individual or population, such as a high risk adult,an elderly adult or an infant. In a specific embodiment, there isprovided the use of an antigen or antigenic preparation thereof and anadjuvant as herein defined in the preparation of an immunogeniccomposition for vaccination of human, in particular a human elderlyadult, against the pathogen from which the antigen in the immunogeniccomposition is derived. Specifically said antigen is an influenza virus,human papilloma virus, Cytomegalovirus, Varicella Zoster virus,Streptococcus pneumoniae, Plasmodium parasite, antigen or antigens orantigenic preparation thereof.

There is also provided a method of vaccination comprising delivery of anantigen or antigenic composition, in particular an influenza virus orHPV, Cytomegalovirus, Varicella Zoster virus, Streptococcus pneumoniae,Plasmodium parasite, or antigenic preparation thereof and an adjuvant ashereinabove defined to an individual or population in need thereof.

In a specific embodiment, the immunogenic composition is capable ofinducing an improved CD4 T-cell immune response against said antigen orantigenic preparation thereof, and in particular is further capable ofinducing either a humoral immune response or an improved B-memory cellresponse or both, compared to that obtained with the un-adjuvantedantigen or antigenic composition. Specifically said CD4 T-cell immuneresponse involves the induction of a cross-reactive CD4 T helperresponse. Specifically said humoral immune response involves theinduction of a cross-reactive humoral immune response.

In a further embodiment, there is provided a method or use ashereinabove defined, for protection against infection or disease causedby a pathogen which is a variant of the pathogen from which the antigenin the immunogenic composition is derived. In another embodiment, thereis provided a method or use as hereinabove defined for protectionagainst infections or disease caused by a pathogen which comprises anantigen which is a variant of that antigen in the immunogeniccomposition. In a specific embodiment, there is provided the use of anantigen, in particular an influenza or HPV, or antigenic preparationthereof in the manufacture of an immunogenic composition forrevaccination of humans previously vaccinated with an immunogeniccomposition comprising an antigen, in particular an influenza or HPV orantigenic preparation thereof, in combination with an adjuvant as hereindescribed.

In a specific embodiment, the composition used for the revaccination mayadditionally contain an adjuvant. In another specific embodiment, theimmunogenic composition for revaccination contains an antigen whichshares common CD4 T-cell epitopes with an antigen or antigeniccomposition used for a previous vaccination. Specifically, theimmunogenic composition for revaccination contains an influenza virus orantigenic preparation thereof which shares common CD4 T-cell epitopeswith the influenza virus or virus antigenic preparation thereof used forthe first vaccination.

In one aspect, the revaccination is made in subjects who have beenvaccinated the previous season against influenza. Typicallyrevaccination is made at least 6 months after the first vaccination,preferably 8 to 14 months after, more preferably at around 10 to 12months after. In another aspect the revaccination is made in subjectswho have been vaccinated with a composition comprising an influenzavirus or antigenic preparation thereof wherein at least one strain isassociated with a pandemic outbreak or has the potential to beassociated with a pandemic outbreak.

In a further aspect of the present invention, there is provided the useof an influenza virus or antigenic preparation thereof from a firstinfluenza strain in the manufacture of an immunogenic composition asherein defined for protection against influenza infections caused by avariant influenza strain.

The invention also relates to a method of vaccination comprisingdelivery of an influenza virus or antigenic preparation thereof and anadjuvant as herein defined.

In another aspect, there is provided a method of vaccination of animmuno-compromised human individual or population such as high riskadults or elderly, comprising administering an influenza immunogeniccomposition comprising an influenza virus or antigenic preparationthereof in combination with an adjuvant as herein defined.

In still another embodiment, the invention provides a method forrevaccinating humans previously vaccinated with an influenza immunogeniccomposition comprising an influenza antigen or antigenic preparationthereof from at least one influenza virus strains in combination with anadjuvant as herein defined, said method comprising administering to saidhuman an immunogenic composition comprising an influenza virus orantigenic preparation thereof, either adjuvanted or un-adjuvanted.

The invention also relates to a method for the preparation of animmunogenic composition comprising combining a saponin adjuvant in theform of a liposome with an influenza virus or antigenic preparationthereof, and optionally with 3D-MPL.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

DESCRIPTION OF FIGURES

FIG. 1—diagrammatic representation of MPL preparation.

FIGS. 2A-2D—Humoral response against various strains of influenzafollowing immunization of ferrets with experimental formulations:Hemagglutination Inhibition Test (GMT+/−IC95) before and afterheterologous priming (H1N1 A/Stockholm/24/90), after immunization (H1N1A/New Caledonia/20/99 (FIG. 2A), H3N2 A/Panama/2007/99 (FIG. 2C) andB/Shangdong/7/97)(FIG. 2B) and after heterologous challenge (H3N2A/Wyoming/3/2003)(FIG. 2D) FIG. 3—Ferret study: Viral titration in nasalwashes after challenge (Day 42) FIG. 4—Mice study: Humoral responseagainst the three vaccine strains of influenza following immunization ofmice with experimental formulations: Hemagglutination Inhibition Test(GMT+/−IC95) 21 days after immunization (H1N1 A/New Caledonia/20/99,H3N2 A/Wyoming/3/2003 and B/Jiangsu/10/2003).

FIG. 5—Mice study: Cell mediated immune response: Flu-specific CD4+ Tcell responses on Day 7 Post-immunization.

FIG. 6—Mice study: CMI for CD4—Pooled strain (all double)—Day 0 and Day21

FIG. 7—GMTs at days 0 and 21 for HI antibodies

FIG. 8: Incidence of local and general symptoms in humans (Total andgrade 3 related) reported during the 7-day follow up period followingimmunisation with adjuvanted influenza virus formulations, comparingadjuvants having two different concentrations of immunostimulants.

FIG. 9: Humoral responses to HPV 16 and 18 L1 in mice followingimmunisation with adjuvanted HPV formulations, comparing adjuvantshaving two different concentrations of immunostimulants

FIG. 10: Cell mediated immune response in mice: Intracellular CytokineStaining—VLP16 and 18 CD4+ T cells following immunisation withadjuvanted HPV formulations, comparing adjuvants having two differentconcentrations of immunostimulants

FIG. 11: Production of Specific B Memory cells following immunisationwith adjuvanted HPV formulations, comparing adjuvants having twodifferent concentrations of immunostimulants

FIG. 12: Preclinical comparison of adjuvanted S. pneumonaie vaccines inmice, comparing adjuvants having two different concentrations ofimmunostimulants.

FIG. 13: Guinea pig Anti-gB ELISA titers following immunisation withadjuvanted Gb vaccine, comparing adjuvants having two differentconcentrations of immunostimulants.

FIG. 14: Guinea Pig Anti CMV neutralizing titers following immunisationwith adjuvanted Gb vaccine, comparing adjuvants having two differentconcentrations of immunostimulants.

FIG. 15: Mice Anti-gB ELISA titers following immunisation with adjuvantgB vaccine.

FIG. 16: Mice anti CMV neutralising titers following immunisation withadjuvanted gB vaccine.

FIG. 17: Mice study: Cell Mediated immunity—CMV specific CD4+ and CD8+cells following re-stimulation with a pool of gB peptides (7 days postsecond immunisation)

FIG. 18: Mice study. Cell Mediated immunity—CMV specific CD4+ cellsfollowing re-stimulation with two different dosages of a pool of gBpeptides (21 days post second immunisation).

FIG. 19: Mice study. Cell Mediated immunity—CMV specific CD8+ cellsfollowing re-stimulation with two different dosages of a pool of gBpeptides (21 days post second immunisation).

FIG. 20: Geometric mean antibody titers (GMT) against Circumsporozoiteprotein CSP following immunization with adjuvanted RTS,S vaccine inmice; comparing adjuvants having immunostimulants at two differentconcentrations.

FIG. 21: Geometric mean antibody titers (GMT) against Hepatitis Bsurface antigen (HBs) following immunization with adjuvanted RTS,Svaccine in mice; comparing adjuvants with immunostimulants at twodifferent concentrations.

FIG. 22: Ex vivo expression of IL-2 and/or IFN gamma by CSP-specific CD4and CD8 T cells following immunization with an adjuvanted RTS,Simmunogenic composition, comparing adjuvants with immunostimulants attwo different concentrations.

FIG. 23: Ex vivo expression of IL-2 and/or IFN gamma by HBs-specific CD4and CD8 T cells following immunization with an adjuvanted RTS,Simmunogenic composition, comparing adjuvants with immunostimulants attwo different concentrations.

FIG. 24: Humoral responses in mice following immunisation withadjuvanted trivalent split influenza vaccine (A/New Caledonia,A/Wyoming,B/Jiangsu), immunostimulants at two different concentrations.

FIG. 25: Cell mediated immune response in mice following immunisationwith adjuvanted trivalent influenza vaccine (A/New Caledonia,A/Wyoming,B/Jiangsu), immunostimulants at two different concentrations.

FIG. 26: Preclinical results in mice comparing VZV gE vaccines adjuvantwith AS01B or AS01E.

FIG. 27: viral nasal wash titres following priming and challenge withinfluenza virus antigens-immunisation with A/New Caledonia,A/Wyoming,B/Jiangsu either plain or adjuvanted with adjuvant compositionscomprising immunostimulants at two different concentrations, in ferrets

FIG. 28: Body temperature monitoring in ferrets following priming andchallenge with influenza antigens. Immunisation with A/NewCaledonia,A/Wyoming, B/Jiangsu either plain or adjuvanted with adjuvantcompositions comprising immunostimulants at two differentconcentrations,

FIG. 29: Anti HI titers for the A strains in the trivalent vaccineformulation following immunisation and challenge with influenza antigenpreparations. Immunisation with A/New Caledonia,A/Wyoming, B/Jiangsueither plain or adjuvanted with adjuvant compositions comprisingimmunostimulants at two different concentrations,

FIG. 30: Anti HI titres for B/Jiangsu and the drift strain used forchallenge following immunisation and challenge with influenza antigenpreparations. Immunisation with A/New Caledonia,A/Wyoming, B/Jiangsueither plain or adjuvanted with adjuvant compositions comprisingimmunostimulants at two different concentrations,

DETAILED DESCRIPTION

The present inventors have discovered that an adjuvant composition whichcomprises a saponin presented in the form of a liposome, and alipopolysaccharide, where each immunostimulant is present at a level ator below 30 μg per human dose can improve immune responses to anantigenic preparation, whilst at the same time having lowerreactogenicity than some of the prior art formulations where theimmunostimulants were present at higher levels per human dose.

The present inventors have further found that an influenza formulationcomprising an influenza virus or antigenic preparation thereof togetherwith an adjuvant comprising a saponin presented in the form of aliposome, and optionally additionally with a lipid A derivative such as3D-MPL, was capable of improving the CD4 T-cell immune response againstsaid antigen or antigenic composition compared to that obtained with theun-adjuvanted virus or antigenic preparation thereof. The formulationsadjuvanted with saponin presented in the form of a liposome areadvantageously used to induce anti-influenza CD4-T cell responsescapable of detection of influenza epitopes presented by MHC class IImolecules. The present applicant has found that it is effective totarget the cell-mediated immune system in order to increaseresponsiveness against homologous and drift influenza strains (uponvaccination and infection).

It is a specific embodiment of the present invention that thecompositions for use in the present invention may be able to provide, inhumans, better sero-protection against influenza followingrevaccination, as assessed by the number of human subjects meeting theinfluenza correlates of protection. Furthermore, it is another specificembodiment that the composition for use in the present invention willalso be able to induce a higher B cell memory response following thefirst vaccination of a human subject, and a higher humoral responsefollowing revaccination, compared to the un-adjuvanted composition.

The adjuvanted influenza compositions according to the invention haveseveral advantages:

-   -   1) An improved immunogenicity: they will allow to restore weak        immune response in the elderly people (over 50 years of age,        typically over 65 years of age) to levels seen in young people        (antibody and/or T cell responses);    -   2) An improved cross-protection profile: increased        cross-protection against variant (drifted) influenza strains;    -   3) They will also allow a reduced antigen dosage to be used for        a similar response, thus ensuring an increased capacity in case        of emergency (pandemics for example).

In another aspect of the invention, the inventors have discovered thatthe adjuvant composition as defined herein demonstrates immunogenicityresults for both antibody production and post-vaccination frequency ofinfluenza-specific CD4 which are equivalent to, or sometimes greaterthan, those generated with non-adjuvanted vaccine. This effect is inparticular of value in the elderly population and can be achieved withan adjuvant as herein defined containing a lower dose ofimmunostimulants. In addition, reactogenicity symptoms showed a trend tobe higher in the group who received the vaccine adjuvanted with thehighest immunostimulants concentration compared to the group whoreceived the adjuvanted vaccine wherein the immunostimulants is at alower concentration.

These findings can be applied to other forms of the same antigens, andto other antigens.

Saponin Adjuvant

The adjuvant composition of the invention comprises a saponin adjuvantpresented in the form of a liposome.

A particularly suitable saponin for use in the present invention is QuilA and its derivatives. Quil A is a saponin preparation isolated from theSouth American tree Quillaja Saponaria Molina and was first described byDalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. für die gesamteVirusforschung, Vol. 44, Springer Verlag, Berlin, p243-254) to haveadjuvant activity. Purified fragments of Quil A have been isolated byHPLC which retain adjuvant activity without the toxicity associated withQuil A (EP 0 362 278), for example QS7 and QS21 (also known as QA7 andQA21). QS-21 is a natural saponin derived from the bark of Quillajasaponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cellsand a predominant IgG2a antibody response and is a preferred saponin inthe context of the present invention.

In a suitable form of the present invention, the saponin adjuvant withinthe immunogenic composition is a derivative of saponaria molina quil A,preferably an immunologically active fraction of Quil A, such as QS-17or QS-21, suitably QS-21. In one embodiment the compositions of theinvention contain the immunologically active saponin fraction insubstantially pure form. Preferably the compositions of the inventioncontain QS21 in substantially pure form, that is to say, the QS21 is atleast 90% pure, for example at least 95% pure, or at least 98% pure.

In a specific embodiment, QS21 is provided in its less reactogeniccomposition where it is quenched with an exogenous sterol, such ascholesterol for example. Several particular forms of less reactogeniccompositions wherein QS21 is quenched with an exogenous cholesterolexist. In a specific embodiment, the saponin/sterol is in the form of aliposome structure (WO 96/33739, Example 1). In this embodiment theliposomes suitably contain a neutral lipid, for examplephosphatidylcholine, which is suitably non-crystalline at roomtemperature, for example eggyolk phosphatidylcholine, dioleoylphosphatidylcholine (DOPC) or dilauryl phosphatidylcholine. Theliposomes may also contain a charged lipid which increases the stabilityof the lipsome-QS21 structure for liposomes composed of saturatedlipids. In these cases the amount of charged lipid is suitably 1-20%w/w, preferably 5-10%. The ratio of sterol to phospholipid is 1-50%(mol/mol), suitably 20-25%.

Suitable sterols include β-sitosterol, stigmasterol, ergosterol,ergocalciferol and cholesterol. In one particular embodiment, theadjuvant composition comprises cholesterol as sterol. These sterols arewell known in the art, for example cholesterol is disclosed in the MerckIndex, 11th Edn., page 341, as a naturally occurring sterol found inanimal fat.

Adjuvant compositions of the invention comprising QS21 and a sterol,cholesterol in particular, show a decreased reactogenicity when comparedto compositions in which the sterol is absent, while the adjuvant effectis maintained. Reactogenicity studies may be assessed according to themethods disclosed in WO 96/33739. The sterol according to the inventionis taken to mean an exogenous sterol, i.e. a sterol which is notendogenous to the organism from which the antigenic preparation is takenbut is added to the antigen preparation or subsequently at the moment offormulation. Typically, the sterol may be added during subsequentformulation of the antigen preparation with the saponin adjuvant, byusing, for example, the saponin in its form quenched with the sterol.Suitably the exogenous sterol is associated to the saponin adjuvant asdescribed in WO 96/33739.

Where the active saponin fraction is QS21, the ratio of QS21:sterol willtypically be in the order of 1:100 to 1:1 (w/w), suitably between 1:10to 1:1 (w/w), and preferably 1:5 to 1:1 (w/w). Suitably excess sterol ispresent, the ratio of QS21:sterol being at least 1:2 (w/w). In oneembodiment, the ratio of QS21:sterol is 1:5 (w/w). The sterol issuitably cholesterol.

Other useful saponins are derived from the plants Aesculus hippocastanumor Gyophilla struthium. Other saponins which have been described in theliterature include Escin, which has been described in the Merck index(12^(th) ed: entry 3737) as a mixture of saponins occuring in the seedof the horse chestnut tree, Lat: Aesculus hippocastanum. Its isolationis described by chromatography and purification (Fiedler,Arzneimittel-Forsch. 4, 213 (1953)), and by ion-exchange resins (Erbringet al., U.S. Pat. No. 3,238,190). Fractions of escin have been purifiedand shown to be biologically active (Yoshikawa M, et al. (Chem PharmBull (Tokyo) 1996 August; 44(8):1454-1464)). Sapoalbin from Gypsophillastruthium (R. Vochten et al., 1968, J. Pharm.Belg., 42, 213-226) hasalso been described in relation to ISCOM production for example.

A key aspect of the present invention is the fact that theimmunologically active saponin, which is preferably QS21, can be used atlower amounts than had previously been thought useful, suitably at below30 μg, for example between 1 and 30 μg, per human dose of theimmunogenic composition.

The invention therefore provides a human dose of an immunogeniccomposition comprising immunologically active saponin, preferably QS21,at a level of 30 μg or less, for example between 1 and 30 μg.

In one embodiment, an immunogenic composition in a volume which issuitable for a human dose which human dose of the immunogeniccomposition comprises QS21 at a level of around 25 μg, for examplebetween 20-30 μg, suitably between 21-29 μg or between 22 and 28 μg orbetween 23 and 27 μg or between 24 and 26 μg, or 25 μg.

In another embodiment, the human dose of the immunogenic compositioncomprises QS21 at a level of around 10 μg per, for example between 5 and15 μg, suitably between 6 and 14 μg, for example between 7 and 13 μg orbetween 8 and 12 μg or between 9 and 11 μg, or 10 μg.

In a further embodiment, the human dose of the immunogenic compositioncomprises QS21 at a level of around 5 μg, for example between 1 and 9μg, or between 2 and 8 μg or suitably between 3 and 7 μg or 4 and 6 μg,or 5 μg.

A suitable amount of QS21 is for example any of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, μg (w/v) per human dose of the immunogenic composition.

By the term “human dose” is meant a dose which is in a volume suitablefor human use. Generally this is between 0.3 and 1.5 ml. In oneembodiment, a human dose is 0.5 ml. In a further embodiment, a humandose is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In afurther embodiment, a human dose is between 1 ml and 1.5 ml. Theinvention is characterised in that each human dose contains 30 μg orless, for example between 1 and 30 μg, of QS21.

The invention further provides an adjuvant composition comprising 30 μgor less, for example between 1 and 30 μg, of QS21. Typically such anadjuvant composition will be in a human dose suitable volume. Where theadjuvant is in a liquid form to be combined with a liquid form of anantigenic composition, the adjuvant composition will be in a human dosesuitable volume which is approximately half of the intended final volumeof the human dose, for example a 360 μl volume for an intended humandose of 0.7 ml, or a 250 μl volume for an intended human dose of 0.5 ml.The adjuvant composition is diluted when combined with the antigencomposition to provide the final human dose of vaccine. The final volumeof such dose will of course vary dependent on the initial volume of theadjuvant composition and the volume of antigen composition added to theadjuvant composition. In an alternative embodiment, liquid adjuvant isused to reconstitute a lyophilised antigen composition. In thisembodiment, the human dose suitable volume of the adjuvant compositionis approximately equal to the final volume of the human dose. The liquidadjuvant composition is added to the vial containing the lyophilisedantigen composition. The final human dose can vary between 0.5 and 1.5ml. In a particular embodiment the human dose is 0.5 ml, in thisembodiment the vaccine composition of the invention will comprise alevel of QS21 at or below 30 μg, for example between 1 and 30 μg, per0.5 ml human dose, furthermore in this embodiment an adjuvantcomposition of the invention will comprise a level of QS21 at or below30 μg, for example between 1 and 30 μg, per 250 μl of adjuvantcomposition, or per 500 μl of adjuvant composition dependent on whetherthe adjuvant composition is intended to be combined with a liquid orlyophilised antigen composition respectively.

Specifically when combined with an influenza antigen, an amount of QS21can be used, for example, at an amount of 1 to 100 μg (w/v) percomposition dose, preferably in an amount of 10 to 50 μg (w/v) percomposition dose. A suitable amount of QS21 is for example any of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 μg (w/v) per composition dose.More preferably, QS21 amount ranges from 25 to 75 μg (w/v) percomposition dose. Usually a composition dose will be ranging from about0.5 ml to about 1 ml. A typical vaccine dose are 0.5 ml, 0.6 ml, 0.7 ml,0.8 ml, 0.9 ml or 1 ml. In a preferred embodiment, a final concentrationof 50 μg of QS21 is contained per ml of vaccine composition, or 25 μgper 0.5 ml vaccine dose. In other preferred embodiments, a finalconcentration of 35.7 μg or 71.4 μg of QS21 is contained per ml ofvaccine composition. Specifically, a 0.5 ml vaccine dose volume contains25 μg or 50 μg of QS21 per dose.

The dose of QS21 is suitably able to enhance an immune response to anantigen in a human. In particular a suitable QS21 amount is that whichimproves the immunological potential of the composition compared to theunadjuvanted composition, or compared to the composition adjuvanted withanother QS21 amount, whilst being acceptable from a reactogenicityprofile.

3D-MPL Adjuvant

The composition further comprises an additional adjuvant which is alipopolysaccharide, suitably a non-toxic derivative of lipid A,particularly monophosphoryl lipid A or more particularly 3-Deacylatedmonophoshoryl lipid A (3D-MPL).

3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A.and is referred throughout the document as MPL or 3D-MPL. see, forexample, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094.3D-MPL primarily promotes CD4+ T cell responses with an IFN-g (Th1)phenotype. 3D-MPL can be produced according to the methods disclosed inGB 2 220 211 A. Chemically it is a mixture of 3-deacylatedmonophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. Preferably inthe compositions of the present invention small particle 3D-MPL is used.Small particle 3D-MPL has a particle size such that it may besterile-filtered through a 0.22 μm filter. Such preparations aredescribed in WO 94/21292.

A key aspect of the present invention is the fact that thelipopolysaccharide, which is preferably 3D-MPL, can be used at loweramounts than had previously been thought useful, suitably at a level of30 μg or less, for example between 1 and 30 μg, per human dose of theimmunogenic composition.

The invention therefore provides a human dose of an immunogeniccomposition comprising lipopolysaccharide, preferably 3D-MPL, at a levelof 30 μg or less, for example between 1 and 30 μg.

In one embodiment, the human dose of the immunogenic compositioncomprises 3D-MPL at a level of around 25 μg, for example between 20-30μg, suitably between 21-29 μg or between 22 and 28 μg or between 23 and27 μg or between 24 and 26 μg, or 25 μg.

In another embodiment, the human dose of the immunogenic compositioncomprises 3D-MPL at a level of around 10 μg, for example between 5 and15 μg, suitably between 6 and 14 μg, for example between 7 and 13 μg orbetween 8 and 12 μg or between 9 and 11 μg, or 10 μg.

In a further embodiment, the human dose of the immunogenic compositioncomprises 3D-MPL at a level of around 5 μg, for example between 1 and 9μg, or between 2 and 8 μg or suitably between 3 and 7 μg or 4 and 6 μg,or 5 μg.

A suitable amount of 3D-MPL is for example any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, μg (w/v) per human dose of the immunogeniccomposition.

In one embodiment, the volume of the human dose is 0.5 ml. In a furtherembodiment, the immunogenic composition is in a volume suitable for ahuman dose which volume is higher than 0.5 ml, for example 0.6, 0.7,0.8, 0.9 or 1 ml. In a further embodiment, the human dose is between 1ml and 1.5 ml. The invention is characterised in that each human dosecontains 30 μg or less, for example between 1 and 30 μg of 3D-MPL.

The invention further provides an adjuvant composition comprising 30 μgor less, for example between 1 and 30 μg, of 3D-MPL. Typically such anadjuvant composition will be in a human dose suitable volume. Where theadjuvant is in a liquid form to be combined with a liquid form of anantigenic composition, the adjuvant composition will be in a human dosesuitable volume which is approximately half of the intended final volumeof the human dose, for example a 360 μl volume for an intended humandose of 0.7 ml, or a 250 μl volume for an intended human dose of 0.5 ml.The adjuvant composition is diluted when combined with the antigencomposition to provide the final human dose of immunogenic composition.The final volume of such dose will of course vary dependent on theinitial volume of the adjuvant composition and the volume of antigencomposition added to the adjuvant composition. In an alternativeembodiment, liquid adjuvant composition is used to reconstitute alyophilised antigen composition. In this embodiment, the human dosesuitable volume of the adjuvant composition is approximately equal tothe final volume of the human dose. The liquid adjuvant composition isadded to the vial containing the lyophilised antigen composition. Thefinal human dose can vary between 0.5 and 1.5 ml. In a particularembodiment the human dose is 0.5 ml, in this embodiment the vaccinecomposition of the invention will comprise a level of 3D-MPL at or below30 μg, for example between 1 and 30 μg, per 0.5 ml human dose,furthermore in this embodiment an adjuvant composition of the inventionwill comprise a level of 3D-MPL at or below 30 μg, for example between 1and 30 μg, per 250 μl of adjuvant composition, or per 500 μl of adjuvantcomposition dependent on whether the adjuvant composition is intended tobe combined with a liquid or lyophilised antigen compositionrespectively.

When the immunogenic composition contains an influenza virus orantigenic preparation thereof, the adjuvant composition which comprisesa saponin in the form of a liposome optionally additionally contains alipid A derivative, particularly monophosphoryl lipid A or moreparticularly 3D-MPL. In this embodiment, 3D-MPL can be used, forexample, at an amount of 1 to 100 μg (w/v) per composition dose,preferably in an amount of 10 to 50 μg (w/v) per composition dose. Asuitable amount of 3D-MPL is for example any of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or 50 μg (w/v) per composition dose. Morepreferably, 3D-MPL amount ranges from 25 to 75 μg (w/v) per compositiondose. Usually a composition dose will be ranging from about 0.5 ml toabout 1 ml. A typical vaccine dose are 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml,0.9 ml or 1 ml. In one embodiment, a final concentration of 50 μg of3D-MPL is contained per ml of vaccine composition, or 25 μg per 0.5 mlvaccine dose. In another embodiment, a final concentration of 35.7 μg or71.4 μg of 3D-MPL is contained per ml of vaccine composition.Specifically, a 0.5 ml vaccine dose volume contains 25 μg or 50 μg of3D-MPL per dose.

The dose of 3D-MPL is suitably able to enhance an immune response to anantigen in a human. In particular a suitable 3D-MPL amount is that whichimproves the immunological potential of the composition compared to theunadjuvanted composition, or compared to the composition adjuvanted withanother MPL amount, whilst being acceptable from a reactogenicityprofile.

Suitable compositions of the invention are those wherein liposomes areinitially prepared without MPL (as described in WO 96/33739), and MPL isthen added, suitably as small particles of below 100 nm particles orparticles that are susceptible to sterile filtration through a 0.22 μmmembrane. The MPL is therefore not contained within the vesicle membrane(known as MPL out). Compositions where the MPL is contained within thevesicle membrane (known as MPL in) also form an aspect of the invention.The antigen can be contained within the vesicle membrane or containedoutside the vesicle membrane. Suitably soluble antigens are outside andhydrophobic or lipidated antigens are either contained inside or outsidethe membrane.

In one embodiment the adjuvant composition of the invention comprisesboth lipopolysaccharide and immunologically active saponin. In aspecific embodiment of the invention, the lipopolysaccharide is 3D-MPLand the immunologically active saponin is QS21. In a further embodimentof the invention, the adjuvant composition consists essentially of alipopolysaccharide and immunologically active saponin in a liposomalformulation. Suitably in one form of this embodiment, the adjuvantcomposition consists essentially of 3D-MPL and QS21, with optionallysterol which is preferably cholesterol.

In a further embodiment of the invention, the adjuvant compositioncomprises in a liposomal formulation lipopolysaccharide andimmunologically active saponin in combination with one or more furtherimmunostimulants or adjuvants. Suitably in one form of this embodimentthe lipopolysaccharide is 3D-MPL and the immunologically active saponinis QS21.

In a specific embodiment, QS21 and 3D-MPL are present in the same finalconcentration per human dose of the immunogenic composition. In oneaspect of this embodiment, a human dose of immunogenic compositioncomprises a final level of 25 μg of 3D-MPL and 25 μg of QS21. In afurther embodiment, a human dose of immunogenic composition comprises afinal level of 10 μg each of MPL and QS21. In a further specificembodiment is provided an adjuvant composition having a volume of 250 μland comprising a level of 25 μg of 3D-MPL and 25 μg of QS21, or 10 μgeach of MPL and QS21.

Antigens that may be used with the adjuvant compositions of the presentinvention include viral, parasitic, bacterial or tumour associatedantigens, for example:

An influenza virus or antigenic preparation thereof for use according tothe present invention, which may be a split influenza virus or splitvirus antigenic preparation thereof. In an alternative embodiment theinfluenza preparation may contain another type of inactivated influenzaantigen, such as inactivated whole virus or purified HA and NA (subunitvaccine), or an influenza virosome. In a still further embodiment, theinfluenza virus may be a live attenuated influenza preparation.

A split influenza virus or split virus antigenic preparation thereof foruse according to the present invention is suitably an inactivated viruspreparation where virus particles are disrupted with detergents or otherreagents to solubilise the lipid envelope. Split virus or split virusantigenic preparations thereof are suitably prepared by fragmentation ofwhole influenza virus, either infectious or inactivated, withsolubilising concentrations of organic solvents or detergents andsubsequent removal of all or the majority of the solubilising agent andsome or most of the viral lipid material. By split virus antigenicpreparation thereof is meant a split virus preparation which may haveundergone some degree of purification compared to the split virus whilstretaining most of the antigenic properties of the split viruscomponents. For example, when produced in eggs, the split virus may bedepleted from egg-contaminating proteins, or when produced in cellculture, the split virus may be depleted from host cell contaminants. Asplit virus antigenic preparation may comprise split virus antigeniccomponents of more than one viral strain. Vaccines containing splitvirus (called ‘influenza split vaccine’) or split virus antigenicpreparations generally contain residual matrix protein and nucleoproteinand sometimes lipid, as well as the membrane envelope proteins. Suchsplit virus vaccines will usually contain most or all of the virusstructural proteins although not necessarily in the same proportions asthey occur in the whole virus.

Alternatively, the influenza virus may be in the form of a whole virusvaccine. This may prove to be an advantage over a split virus vaccinefor a pandemic situation as it avoids the uncertainty over whether asplit virus vaccine can be successfully produced for a new strain ofinfluenza virus. For some strains the conventional detergents used forproducing the split virus can damage the virus and render it unusable.Although there is always the possibility to use different detergentsand/or to develop a different process for producing a split vaccine,this would take time, which may not be available in a pandemicsituation. In addition to the greater degree of certainty with a wholevirus approach, there is also a greater vaccine production capacity thanfor split virus since considerable amounts of antigen are lost duringadditional purification steps necessary for preparing a suitable splitvaccine.

In another embodiment, the influenza virus preparation is in the form ofa purified sub-unit influenza vaccine. Sub-unit influenza vaccinesgenerally contain the two major envelope proteins, HA and NA, and mayhave an additional advantage over whole virion vaccines as they aregenerally less reactogenic, particularly in young vaccinees. Sub-unitvaccines can be produced either recombinantly or purified from disruptedviral particles.

In another embodiment, the influenza virus preparation is in the form ofa virosome. Virosomes are spherical, unilamellar vesicles which retainthe functional viral envelope glycoproteins HA and NA in authenticconformation, intercalated in the virosomes' phospholipids bilayermembrane.

Said influenza virus or antigenic preparation thereof may be egg-derivedor cell-culture derived.

For example, the influenza virus antigen or antigenic preparationsthereof according to the invention may be derived from the conventionalembryonated egg method, by growing influenza virus in eggs and purifyingthe harvested allantoic fluid. Eggs can be accumulated in large numbersat short notice. Alternatively, they may be derived from any of the newgeneration methods using cell or cell culture to grow the virus orexpress recombinant influenza virus surface antigens. Suitable cellsubstrates for growing the virus include for example dog kidney cellssuch as MDCK or cells from a clone of MDCK, MDCK-like cells, monkeykidney cells such as AGMK cells including Vero cells, suitable pig celllines, or any other mammalian cell type suitable for the production ofinfluenza virus for vaccine purposes. Suitable cell substrates alsoinclude human cells e.g. MRC-5 cells. Suitable cell substrates are notlimited to cell lines; for example primary cells such as chicken embryofibroblasts and avian cell lines are also included.

The influenza virus antigen or antigenic preparation thereof may beproduced by any of a number of commercially applicable processes, forexample the split flu process described in patent no. DD 300 833 and DD211 444, incorporated herein by reference. Traditionally split flu wasproduced using a solvent/detergent treatment, such as tri-n-butylphosphate, or diethylether in combination with Tween™ (known as“Tween-ether” splitting) and this process is still used in someproduction facilities. Other splitting agents now employed includedetergents or proteolytic enzymes or bile salts, for example sodiumdeoxycholate as described in patent no. DD 155 875, incorporated hereinby reference. Detergents that can be used as splitting agents includecationic detergents e.g. cetyl trimethyl ammonium bromide (CTAB), otherionic detergents e.g. laurylsulfate, taurodeoxycholate, or non-ionicdetergents such as the ones described above including Triton X-100 (forexample in a process described in Lina et al, 2000, Biologicals 28,95-103) and Triton N-101, or combinations of any two or more detergents.

The preparation process for a split vaccine may include a number ofdifferent filtration and/or other separation steps such asultracentrifugation, ultrafiltration, zonal centrifugation andchromatography (e.g. ion exchange) steps in a variety of combinations,and optionally an inactivation step eg with heat, formaldehyde orβ-propiolactone or U.V. which may be carried out before or aftersplitting. The splitting process may be carried out as a batch,continuous or semi-continuous process. A preferred splitting andpurification process for a split immunogenic composition is described inWO 02/097072.

Preferred split flu vaccine antigen preparations according to theinvention comprise a residual amount of Tween 80 and/or Triton X-100remaining from the production process, although these may be added ortheir concentrations adjusted after preparation of the split antigen.Preferably both Tween 80 and Triton X-100 are present. The preferredranges for the final concentrations of these non-ionic surfactants inthe vaccine dose are:

Tween 80: 0.01 to 1%, more preferably about 0.1% (v/v)

Triton X-100: 0.001 to 0.1 (% w/v), more preferably 0.005 to 0.02%(w/v).

In a specific embodiment, the final concentration for Tween 80 rangesfrom 0.025%-0.09% w/v. In another specific embodiment, the antigen isprovided as a 2 fold concentrated mixture, which has a Tween 80concentration ranging from 0.025%-0.2% (w/v) and has to be diluted twotimes upon final formulation with the adjuvanted (or the buffer in thecontrol formulation).

In another specific embodiment, the final concentration for Triton X-100ranges from 0.004%-0.017% w/v. In another specific embodiment, theantigen is provided as a 2 fold concentrated mixture, which has a TritonX-100 concentration ranging from 0.005%-0.034% (w/v) and has to bediluted two times upon final formulation with the adjuvanted (or thebuffer in the control formulation).

Preferably the influenza preparation is prepared in the presence of lowlevel of thiomersal, or preferably in the absence of thiomersal.Preferably the resulting influenza preparation is stable in the absenceof organomercurial preservatives, in particular the preparation containsno residual thiomersal. In particular the influenza virus preparationcomprises a haemagglutinin antigen stabilised in the absence ofthiomersal, or at low levels of thiomersal (generally 5 μg/ml or less).Specifically the stabilization of B influenza strain is performed by aderivative of alpha tocopherol, such as alpha tocopherol succinate (alsoknown as vitamin E succinate, i.e. VES). Such preparations and methodsto prepare them are disclosed in WO 02/097072.

A preferred composition contains three inactivated split virion antigensprepared from the WHO recommended strains of the appropriate influenzaseason.

Preferably the influenza virus or antigenic preparation thereof and theadjuvant according to the invention are contained in the same container.It is referred to as ‘one vial approach’. Preferably the vial is apre-filled syringe. In an alternative embodiment, the influenza virus orantigenic preparation thereof and adjuvant according to the inventionare contained in separate containers or vials and admixed shortly beforeor upon administration into the subject. It is referred to as ‘two vialsapproach’. By way of example, when the vaccine is a 2 components vaccinefor a total dose volume of 0.7 ml, the concentrated antigens (forexample the concentrated trivalent inactivated split virion antigens)are presented in one vial (335 μl) (antigen container) and a pre-filledsyringe contains the adjuvant (360 μl) (adjuvant container). At the timeof injection, the content of the vial containing the concentratedtrivalent inactivated split virion antigens is removed from the vial byusing the syringe containing the adjuvant followed by gentle mixing ofthe syringe. Prior to injection, the used needle is replaced by anintramuscular needle and the volume is corrected to 530 μl. In thisexample, one dose of the reconstituted adjuvanted influenza vaccinecandidate corresponds to 530 μl.

In one aspect of the invention, where there is a multivalentcomposition, then at least one influenza strain in said multivalentimmunogenic composition as herein defined is associated with a pandemicoutbreak or have the potential to be associated with a pandemicoutbreak. Such strain may also be referred to as ‘pandemic strains’ inthe text below. In particular, when the vaccine is a multivalent vaccinesuch as a bivalent, or a trivalent or a quadrivalent vaccine, at leastone strain is associated with a pandemic outbreak or has the potentialto be associated with a pandemic outbreak. Suitable strains are, but notlimited to: H5N1, H9N2, H7N7, and H2N2.

Said influenza virus or antigenic preparation thereof is suitablymultivalent such as bivalent or trivalent or quadrivalent. Preferablythe influenza virus or antigenic preparation thereof is trivalent orquadrivalent, having an antigen from three different influenza strains,at least one strain being associated with a pandemic outbreak or havingthe potential to be associated with a pandemic outbreak.

The features of an influenza virus strain that give it the potential tocause a pandemic outbreak are: it contains a new haemagglutinin comparedto the haemagglutinin in the currently circulating strains; it iscapable of being transmitted horizontally in the human population; andit is pathogenic for humans. A new haemagglutinin may be one which hasnot been evident in the human population for an extended period of time,probably a number of decades, such as H2. Or it may be a haemagglutininthat has not been circulating in the human population before, forexample H5, H9, H7 or H6 which are found in birds. In either case themajority, or at least a large proportion of, or even the entirepopulation has not previously encountered the antigen and isimmunologically naïve to it.

Certain parties are generally at an increased risk of becoming infectedwith influenza in a pandemic situation. The elderly, the chronically illand small children are particularly susceptible but many young andapparently healthy people are also at risk. For H2 influenza, the partof the population born after 1968 is at an increased risk. It isimportant for these groups to be protected effectively as soon aspossible and in a simple way.

Another group of people who are at increased risk are travelers. Peopletravel more today than ever before and the regions where most newviruses emerge, China and South East Asia, have become popular traveldestinations in recent years. This change in travel patterns enables newviruses to reach around the globe in a matter of weeks rather thanmonths or years.

Thus for these groups of people there is a particular need forvaccination to protect against influenza in a pandemic situation or apotential pandemic situation. Suitable strains are, but not limited to:H5N1, H9N2, H7N7, and H2N2.

Optionally the composition may contain more than three valencies, forexample three non pandemic strains plus a pandemic strain. Alternativelythe composition may contain three pandemic strains. Preferably thecomposition contains three pandemic strains.

Also examples of antigens for the immunogenic composition of theinvention are Streptococcal antigens such as from Group A Streptococcus,or Group B Streptococcus, but most preferably from Streptococcuspneumoniae. At least one protein and/or at least one saccharide antigenis most preferably used. The at least one Streptococcus pneumoniaeprotein antigen(s) is most preferably selected from the group consistingof: pneumolysin, PspA or transmembrane deletion variants thereof, PspCor transmembrane deletion variants thereof, PsaA or transmembranedeletion variants thereof, glyceraldehyde-3-phosphate dehydrogenase,CbpA or transmembrane deletion variants thereof, PhtA, PhtD, PhtB, PhtE,SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, orimmunologically functional equivalent thereof (for instance fusions ofdomains of the above proteins, for instance the PhtDE fusion proteinsdescribed in WO01/98334 and WO 03/54007).

Certain compositions are described in WO 00/56359 and WO 02/22167 and WO02/22168 (incorporated by reference herein).

The antigen may comprise capsular saccharide antigens (preferablyconjugated to a carrier protein), wherein the saccharides (mostpreferably polysaccharides) are derived from at least four serotypes ofpneumococcus. In one embodiment the four serotypes include 6B, 14, 19Fand 23F. In a further embodiment, at least 7 serotypes are included inthe composition, for example those derived from serotypes 4, 6B, 9V, 14,18C, 19F, and 23F. In a further embodiment, at least 10 serotypes areincluded in the composition, for example the composition in oneembodiment includes capsular saccharides derived from serotypes 1, 4, 5,6B, 7F, 9V, 14, 18C, 19F and 23F (preferably conjugated to a carrierprotein). In another embodiment, the immunogenic composition comprisescapsular saccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14,18C, 19F and 23F In a preferred embodiment of the invention at least 13saccharide antigens (preferably conjugated to a carrier protein) areincluded, although further saccharide antigens, for example 23 valent(such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated bythe invention.

Although the above saccharides are advantageously in their full-length,native polysaccharide form, it should be understood that size-reducedpolysaccharides may also be used which are still immunogenic (see forexample EP 497524 and 497525) if necessary when coupled to a proteincarrier.

For the prevention/amelioration of pneumonia in the elderly (+55 years)population and Otitis media in Infants (up to 18 months) and toddlers(typically 18 months to 5 years), it is a preferred embodiment of theinvention to combine a multivalent Streptococcus pneumonia saccharide asherein described with a Streptococcus pneumoniae protein preferablyselected from the group of proteins listed above. A combination ofpneumococcal proteins may also be advantageously utilised as describedbelow.

Pneumococcal Proteins

Streptococcus pneumoniae antigens are preferably selected from the groupconsisting of: a protein from the polyhistidine triad family (Pht), aprotein from the Lyt family, a choline binding protein, proteins havingan LPXTG motif (where X is any amino acid), proteins having a Type IISignal sequence motif of LXXC (where X is any amino acid), and proteinshaving a Type I Signal sequence motif. Preferred examples within thesecategories (or motifs) are the following proteins (or truncate orimmunologically functional equivalent thereof):

The Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB,PhtD, and PhtE. The family is characterised by a lipidation sequence,two domains separated by a proline-rich region and several histidinetriads, possibly involved in metal or nucleoside binding or enzymaticactivity, (3-5) coiled-coil regions, a conserved N-terminus and aheterogeneous C terminus. It is present in all strains of pneumococcitested. Homologous proteins have also been found in other Streptococciand Neisseria. Preferred members of the family comprise PhtA, PhtB andPhtD. More preferably, it comprises PhtA or PhtD. It is understood,however, that the terms Pht A, B, D, and E refer to proteins havingsequences disclosed in the citations below as well asnaturally-occurring (and man-made) variants thereof that have a sequencehomology that is at least 90% identical to the referenced proteins.Preferably it is at least 95% identical and most preferably it is 97%identical.

With regards to the Pht proteins, PhtA is disclosed in WO 98/18930, andis also referred to Sp36. As noted above, it is a protein from thepolyhistidine triad family and has the type II signal motif of LXXC.

PhtD is disclosed in WO 00/37105, and is also referred to Sp036D. Asnoted above, it also is a protein from the polyhistidine triad familyand has the type II LXXC signal motif. PhtB is disclosed in WO 00/37105,and is also referred to Sp036B. Another member of the PhtB family is theC3-Degrading Polypeptide, as disclosed in WO 00/17370. This protein alsois from the polyhistidine triad family and has the type II LXXC signalmotif. A preferred immunologically functional equivalent is the proteinSp42 disclosed in WO 98/18930. A PhtB truncate (approximately 79 kD) isdisclosed in WO99/15675 which is also considered a member of the PhtXfamily.

PhtE is disclosed in WO00/30299 and is referred to as BVH-3.

SpsA is a Choline binding protein (Cbp) disclosed in WO 98/39450.

The Lyt family is membrane associated proteins associated with celllysis. The N-terminal domain comprises choline binding domain(s),however the Lyt family does not have all the features found in thecholine binding protein family (Cbp) family noted below and thus for thepresent invention, the Lyt family is considered distinct from the Cbpfamily. In contrast with the Cbp family, the C-terminal domain containsthe catalytic domain of the Lyt protein family. The family comprisesLytA, B and C. With regards to the Lyt family, LytA is disclosed inRonda et al., Eur J Biochem, 164:621-624 (1987). LytB is disclosed in WO98/18930, and is also referred to as Sp46. LytC is also disclosed in WO98/18930, and is also referred to as Sp91. A preferred member of thatfamily is LytC.

Another preferred embodiment are Lyt family (particularly LytA)truncates wherein “Lyt” is defined above and “truncates” refers toproteins lacking 50% or more of the Choline binding region. Preferablysuch proteins lack the entire choline binding region.

Sp125 is an example of a pneumococcal surface protein with the Cell WallAnchored motif of LPXTG (where X is any amino acid). Any protein withinthis class of pneumococcal surface protein with this motif has beenfound to be useful within the context of this invention, and istherefore considered a further protein of the invention. Sp125 itself isdisclosed in WO 98/18930, and is also known as ZmpB—a zincmetalloproteinase.

Sp101 is disclosed in WO 98/06734 (where it has the reference # y85993.It is characterised by a Type I signal sequence.

Sp133 is disclosed in WO 98/06734 (where it has the reference # y85992.It is also characterised by a Type I signal sequence.

Sp128 and Sp130 are disclosed in WO 00/76540.

The proteins used in the present invention are preferably selected fromthe group PhtD, PhtA and PhtE, or a combination of 2 or all 3 of theseproteins (i.e. PhtA+D, A+E, D+E or A+D+E). Further pneumococcal proteinantigens that may be included are one or more from the group consistingof: pneumolysin (also referred to as Ply; preferably detoxified bychemical treatment or mutation) [WO 96/05859, WO 90/06951, WO 99/03884],PsaA and transmembrane deletion variants thereof (Berry & Paton, InfectImmun 1996 December; 64(12):5255-62), PspA and transmembrane deletionvariants thereof (U.S. Pat. No. 5,804,193, WO 92/14488, WO 99/53940),PspC and transmembrane deletion variants thereof (WO 97/09994, WO99/53940), a member of the Choline binding protein (Cbp) family [e.g.CbpA and transmembrane deletion variants thereof (WO 97/41151; WO99/51266)], Glyceraldehyde-3-phosphate-dehydrogenase (Infect. Immun.1996 64:3544), HSP70 (WO 96/40928), PcpA (Sanchez-Beato et al. FEMSMicrobiol Lett 1998, 164:207-14), M like protein (SB patent applicationNo. EP 0837130), and adhesin 18627 (SB Patent application No. EP0834568). The present invention also encompasses immunologicallyfunctional equivalents or truncates of such proteins (as defined above).

Concerning the Choline Binding Protein family, members of that familywere originally identified as pneumococcal proteins that could bepurified by choline-affininty chromatography. All of the choline-bindingproteins are non-covalently bound to phosphorylcholine moieties of cellwall teichoic acid and membrane-associated lipoteichoic acid.Structurally, they have several regions in common over the entirefamily, although the exact nature of the proteins (amino acid sequence,length, etc.) can vary. In general, choline binding proteins comprise anN terminal region (N), conserved repeat regions (R1 and/or R2), aproline rich region (P) and a conserved choline binding region (C), madeup of multiple repeats, that comprises approximately one half of theprotein. As used in this application, the term “Choline Binding Proteinfamily (Cbp)” is selected from the group consisting of Choline BindingProteins as identified in WO 97/41151, PbcA, SpsA, PspC, CbpA, CbpD, andCbpG. CbpA is disclosed in WO 97/41151. CbpD and CbpG are disclosed inWO 00/29434. PspC is disclosed in WO 97/09994. PbcA is disclosed in WO98/21337. Preferably the Choline Binding Proteins are selected from thegroup consisting of CbpA, PbcA, SpsA and PspC.

If a Cbp is the further protein utilised it may be a Cbp truncatewherein “Cbp” is defined above and “truncate” refers to proteins lacking50% or more of the Choline binding region (C). Preferably such proteinslack the entire choline binding region. More preferably, the suchprotein truncates lack (i) the choline binding region and (ii) a portionof the N-terminal half of the protein as well, yet retain at least onerepeat region (R1 or R2). More preferably still, the truncate has 2repeat regions (R1 and R2). Examples of such preferred embodiments areNR1×R2, R1×R2, NR1×R2P and R1×R2P as illustrated in WO99/51266 orWO99/51188, however, other choline binding proteins lacking a similarcholine binding region are also contemplated within the scope of thisinvention.

Cbp truncate-Lyt truncate chimeric proteins (or fusions) may also beused in the composition of the invention. Preferably this comprisesNR1×R2 (or R1×R2 or NR1×R2P or R1×R2P) of Cbp and the C-terminal portion(Cterm, i.e., lacking the choline binding domains) of Lyt (e.g.,LytCCterm or Sp91Cterm). More preferably Cbp is selected from the groupconsisting of CbpA, PbcA, SpsA and PspC. More preferably still, it isCbpA. Preferably, Lyt is LytC (also referred to as Sp91).

A PspA or PsaA truncate lacking the choline binding domain (C) andexpressed as a fusion protein with Lyt may also be used. Preferably, Lytis LytC.

In a pneumococcal composition it is possible to combine differentpneumococcal proteins of the invention. Preferably the combination ofproteins of the invention are selected from 2 or more (3 or 4) differentcategories such as proteins having a Type II Signal sequence motif ofLXXC (where X is any amino acid, e.g., the polyhistidine triad family(Pht)), choline binding proteins (Cbp), proteins having a Type I Signalsequence motif (e.g., Sp101), proteins having a LPXTG motif (where X isany amino acid, e.g., Sp128, Sp130), toxins (e.g., Ply), etc. Preferredexamples within these categories (or motifs) are the proteins mentionedabove, or immunologically functional equivalents thereof. Toxin+Pht,toxin+Cbp, Pht+Cbp, and toxin+Pht+Cbp are preferred categorycombinations.

Preferred beneficial combinations include, but are not limited to,PhtD+NR1×R2, PhtD+NR1×R2-Sp91Cterm chimeric or fusion proteins,PhtD+Ply, PhtD+Sp128, PhtD+PsaA, PhtD+PspA, PhtA+NR1×R2,PhtA+NR1×R2-Sp91Cterm chimeric or fusion proteins, PhtA+Ply, PhtA+Sp128,PhtA+PsaA, PhtA+PspA, NR1×R2+LytC, NR1×R2+PspA, NR1×R2+PsaA,NR1×R2+Sp128, R1×R2+LytC, R1×R2+PspA, R1×R2+PsaA, R1×R2+Sp128,R1×R2+PhtD, R1×R2+PhtA. Preferably, NR1×R2 (or R1×R2) is from CbpA orPspC. More preferably it is from CbpA.

A particularly preferred combination of pneumococcal proteins comprisesPly (or a truncate or immunologically functional equivalentthereof)+PhtD (or a truncate or immunologically functional equivalentthereof) optionally with NR1×R2 (or R1×R2 or NR1×R2P or R1×R2P).Preferably, NR1×R2 (or R1×R2 or NR1×R2P or R1×R2P) is from CbpA or PspC.More preferably it is from CbpA.

The antigen may be a pneumococcus saccharide conjugate comprisingpolysaccharide antigens derived from at least four serotypes, preferablyat least seven serotypes, more preferably at least ten serotypes, and atleast one, but preferably 2, 3, or 4, Streptococcus pneumoniae proteinspreferably selected from the group of proteins described above.Preferably one of the proteins is PhtD (or an immunologically functionalequivalent thereof) and/or Ply (or an immunologically functionalequivalent thereof).

A problem associated with the polysaccharide approach to vaccination, isthe fact that polysaccharides per se are poor immunogens. To overcomethis, saccharides may be conjugated to protein carriers, which providebystander T-cell help. It is preferred, therefore, that the saccharidesutilised in the invention are linked to such a protein carrier. Examplesof such carriers which are currently commonly used for the production ofsaccharide immunogens include the Diphtheria and Tetanus toxoids (DT, DTCRM197 and TT respectively), Keyhole Limpet Haemocyanin (KLH), OMPC fromN. meningitidis, and the purified protein derivative of Tuberculin(PPD).

A preferred carrier for the pneumococcal saccharide based immunogeniccompositions (or vaccines) is protein D from Haemophilus influenzae (EP594610-B), or fragments thereof. Fragments suitable for use includefragments encompassing T-helper epitopes. In particular a protein Dfragment will preferably contain the N-terminal 1/3 of the protein. Aprotein D carrier is useful as a carrier in compositions where multiplepneumococcal saccharide antigens are conjugated. One or morepneumococcal saccharides in a combination may be advantageouslyconjugated onto protein D.

A further preferred carrier for the pneumococcal saccharide is thepneumococcal protein itself (as defined above in section “PneumococcalProteins of the invention”).

The saccharide may be linked to the carrier protein by any known method(for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al.,U.S. Pat. No. 4,474,757). Preferably, CDAP conjugation is carried out(WO 95/08348).

Preferably the protein:saccharide (weight:weight) ratio of theconjugates is 0.3:1 to 1:1, more preferably 0.6:1 to 0.8:1, and mostpreferably about 0.7:1.

Particularly preferred compositions of the invention comprise one ormore conjugated pneumococcal saccharides, and one or more pneumococcalproteins of the invention

In addition, pneumococcal saccharides and proteins can be stably storedas bulk components adsorbed onto aluminium phosphate in a liquid form.

In another aspect of the invention, the vaccine composition may comprisea human cytomegalovirus (HCMV) antigen. HCMV is a human DNA virusbelonging to the family of herpes viruses, and is a major cause ofcongenital defects in newborns and also causes serious medicalconditions in immunocompromised patients. Clinical disease causes avariety of symptoms including fever, hepatitis, pneumonititis andinfectious mononucleosis.

In one embodiment, the HCMV antigen is a chimeric fusion protein or animmunogenic derivative thereof comprising a portion of an HCMVglycoprotein fused to a portion of an HSV glycoprotein. The HCMVglycoprotein is typically gB, and the HSV glycoprotein is typically gD,in particular HSV type 2 gD (gD2). The fusion is typically between anamino acid in the N-terminal part of a portion of the HCMV gB proteinand an amino acid at the C terminus of a portion of the HSV gD protein.Both the HCMV gB protein and the HSV gD protein components of the fusionprotein may lack a membrane anchor domain.

The portion of the HCMV gB protein may comprise a non-cleavable form ofHCMV gB. Suitably this is achieved by changing one or more amino acidsat a cleavage site of the protein, for example by exchanging Arg458 andArg459 for Glu and Thr, respectively. The portion of the HSV protein maycomprise the signal sequence of gD2 (amino acids 1 to 25) and optionallyamino acids 26 to 52 of gD2 and/or the sequence from gD2 which isPEDSALLEDPED (SEQ ID NO 1) or functionally equivalents thereof, whichmay be shorter or longer. Further sequences from HSV gD may be added tothe fusion protein, for example at the C terminus of the HCMV gBprotein.

In one embodiment, the fusion protein comprises amino acids 1 to 52 ofthe HSV gD2 protein fused to residues 28 to 685 of the HCMV gB protein.Such a fusion protein is designated HCMV gB685*. In a furtherembodiment, the amino acid sequence PEDSALLEDPED, (SEQ ID NO 1), whichis derived from an internal gD2 sequence, may be included at the Cterminal end of the protein HCMV gB685* to produce the proteindesignated HCMV gB685**. These specific fusion proteins are described inmore detail in WO 95/31555.

Another immunogen suitable for use as an HCMV vaccine is pp65, an HCMVmatrix protein as described in WO 94/00150 (City of Hope).

In a further embodiment of the present invention, immunogeniccompositions contain an antigen or antigenic preparation derived fromthe Human Papilloma Virus (HPV) considered to be responsible for genitalwarts (HPV 6 or HPV 11 and others), and/or the HPV viruses responsiblefor cervical cancer (HPV16, HPV18 and others).

In one embodiment the forms of genital wart prophylactic, ortherapeutic, compositions comprise L1 particles or capsomers, and fusionproteins comprising one or more antigens selected from the HPV proteinsE1, E2, E5 E6, E7, L1, and L2.

In one embodiment the forms of fusion protein are: L2E7 as disclosed inWO 96/26277, and proteinD(1/3)-E7 disclosed in GB 9717953.5(PCT/EP98/05285).

A preferred HPV cervical infection or cancer, prophylactic ortherapeutic composition may comprise HPV 16 or 18 antigens. For example,L1 or L2 antigen monomers, or L1 or L2 antigens presented together as avirus like particle (VLP) or the L1 protein alone presented in a VLP orcaposmer structure. Such antigens, virus like particles and capsomersare per se known. See for example WO94/00152, WO94/20137, WO94/05792,and WO93/02184.

Additional early proteins may be included alone or as fusion proteinssuch as E7, E2 or preferably E5 for example; particularly preferredembodiments of this includes a VLP comprising L1 E7 fusion proteins (WO96/11272).

In one embodiment the HPV 16 antigens comprise the early proteins E6 orE7 in fusion with a protein D carrier to form Protein D-E6 or E7 fusionsfrom HPV 16, or combinations thereof; or combinations of E6 or E7 withL2 (WO 96/26277).

Alternatively the HPV 16 or 18 early proteins E6 and E7, may bepresented in a single molecule, preferably a Protein D-E6/E7 fusion.Such a composition may optionally contain either or both E6 and E7proteins from HPV 18, preferably in the form of a Protein D-E6 orProtein D-E7 fusion protein or Protein D E6/E7 fusion protein.

The composition of the present invention may additionally compriseantigens from other HPV types, preferably from HPV 31 or 33.

Oncogenic HPV types include HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,66 and 68. Thus the composition of the present invention may compriseantigens from one or more of these HPV types, in addition to HPV 16and/or HPV 18.

HPV L1 VLPs or capsomers useful in the invention may comprise or consistof full length L1 or an immunogenic fragment of L1. Where the VLP orcapsomer comprises or consists of an immunogenic fragment of L1, thensuitable immunogenic fragments of HPV L1 include truncations, deletions,substitution, or insertion mutants of L1. Such immunogenic fragments aresuitably capable of raising an immune response, said immune responsebeing capable of recognising an L1 protein such as a virus likeparticle, from the HPV type from which the L1 protein was derived.

Suitable immunogenic L1 fragments include truncated L1 proteins. In oneaspect the truncation removes a nuclear localisation signal. In anotheraspect the truncation is a C terminal truncation. In a further aspectthe C terminal truncation removes fewer than 50 amino acids, such asfewer than 40 amino acids. Where the L1 is from HPV 16 then in anotheraspect the C terminal truncation removes 34 amino acids from HPV 16 L1.Where the L1 is from HPV 18 then in a further aspect the C terminaltruncation removes 35 amino acids from HPV 18 L1.

Suitable truncated HPV 16 and 18 L1 sequences are given in WO 06/114312.

The HPV 16 sequence may also be that disclosed in WO9405792 or U.S. Pat.No. 6,649,167, for example, suitably truncated. Suitable truncates aretruncated at a position equivalent to that discussed above, as assessedby sequence comparison.

An alternative HPV 18 sequence is disclosed in WO9629413, which may besuitably truncated. Suitable truncates are truncated at a positionequivalent to that described above, as assessed by sequence comparison.

Other HPV 16 and HPV 18 sequences are well known in the art and may besuitable for use in the present invention.

Where there is an L1 protein from another HPV type then C terminaltruncations corresponding to those made for HPV 16 and HPV 18 may beused, based upon DNA or protein sequence alignments. Suitabletruncations of HPV 31 and 45 L1 proteins are given in WO 06/114312.

Suitable truncations of, for example, HPV 31, 33, 35, 39, 45, 51, 52,56, 58, 59, 66 and 68 may also be made, in one aspect removingequivalent C terminal portions of the L1 protein to those describedabove, as assessed by sequence alignment.

The L1 protein or immunogenic fragment of the invention may optionallybe in the form of a fusion protein, such as the fusion of the L1 proteinwith L2 or an early protein.

The HPV L1 protein is suitably in the form of a capsomer or virus likeparticle (VLP). In one aspect HPV VLPs may be used in the presentinvention. HPV VLPs and methods for the production of VLPs are wellknown in the art. VLPs typically are constructed from the L1 andoptionally L2 structural proteins of the virus, see for exampleWO9420137, U.S. Pat. No. 5,985,610, WO9611272, U.S. Pat. No.6,599,508B1, U.S. Pat. No. 6,361,778B1, EP 595935. Any suitable HPV VLPmay be used in the present invention which provides cross protection,such as an L1 or L1+L2 VLP. Suitably the VLP is an L1-only VLP.

The composition of the invention may contain a combination of HPV 16 L1VLPs and HPV 18 L1 VLPs, or a combination of HPV L1 VLPs from HPV 16,18, 31 and 45, or larger combinations, and includes HPV 16 and 18 or HPV16, 18, 31 and 45 L1 VLPs, or large combinations, wherein the L1 isoptionally truncated as described herein.

In a particular embodiment of the invention, one or more additionalantigens from cancer-causing HPV types are used with HPV 16 and/or 18antigens, the antigens being selected from the following HPV types: HPV31, 45, 33, 58 and 52. As described herein, the antigen may in each casebe L1 for example in the form of L1 VLPs or capsomers. Thus HPV antigensfor use in the compositions, methods and uses described herein maycomprise or consist of L1 VLPs or capsomers from HPV 16, 18, 31, 45, 33,58 and 52. The L1 VLPs may be L1-only VLPs or in combination withanother antigen such as L2 in L1+L2 VLPs. The L1 protein may suitably betruncated as described herein.

VLP formation can be assessed by standard techniques such as, forexample, electron microscopy and dynamic laser light scattering.

The VLP may comprise full length L1 protein. In one aspect the L1protein used to form the VLP is a truncated L1 protein, as describedabove.

VLPs may be made in any suitable cell substrate such as yeast cells orinsect cells e.g. in a baculovirus expression system, and techniques forpreparation of VLPs are well known in the art, such as WO9913056, U.S.64/694,561, U.S. 62/617,6561 and U.S. Pat. No. 6,245,568, and referencestherein, the entire contents of which are hereby incorporated byreference.

VLPS may be made by disassembly and reassembly techniques, which canprovide for more stable and/or homogeneous papillomavirus VLPs. Forexample, McCarthy et al, 1998 “Quantitative Disassembly and Reassemblyof Human Papillomavirus Type 11 Virus like Particles in Vitro” J.Virology 72(1):33-41, describes the disassembly and reassembly ofrecombinant L1 HPV 11 VLPs purified from insect cells in order to obtaina homogeneous preparation of VLP's. WO9913056 and U.S. Pat. No.6,245,568 also describe disassembly/reassembly processes for making HPVVLPs.

In one aspect HPV VLPS are made as described WO9913056 or U.S. Pat. No.6,245,568

Compositions of the present invention may comprise antigens or antigenicpreparations derived from parasites that cause Malaria such asPlasmodium falciparum or Plasmodium vivax. For example, possibleantigens derived from Plasmodium falciparum include circumsporozoiteprotein (CS protein), RTS,S MSP1, MSP3, LSA1, LSA3, AMA1 and TRAP. RTSis a hybrid protein comprising substantially all the C-terminal portionof the circumsporozoite (CS) protein of P.falciparum linked via fouramino acids of the preS2 portion of Hepatitis B surface antigen to thesurface (S) antigen of hepatitis B virus. Its full structure isdisclosed in the International Patent Application No. PCT/EP92/02591,published under Number WO 93/10152 claiming priority from UK patentapplication No. 9124390.7. When expressed in yeast RTS is produced as alipoprotein particle, and when it is co-expressed with the S antigenfrom HBV it produces a mixed particle known as RTS,S. TRAP antigens aredescribed in the International Patent Application No. PCT/GB89/00895,published under WO 90/01496. A preferred embodiment of the presentinvention is a Malaria vaccine wherein the antigenic preparationcomprises a combination of the RTS,S and TRAP antigens. Other plasmodiaantigens that are likely candidates to be components of a multistageMalaria vaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1,RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1,Pfs25, Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and their analogues inPlasmodium spp. One embodiment of the present invention is a compositioncomprising RTS, S or CS protein or a fragment thereof such as the CSportion of RTS, S in combination with one or more further malarialantigens which may be selected for example from the group consisting ofMSP1, MSP3, AMA1, LSA1 or LSA3. Possible antigens from P. vivax includecircumsporozoite protein (CS protein) and Duffy antigen binding proteinand fragments thereof, such as PvRII (see eg WO02/12292).

Other possible antigens that may be used in the immunogenic compositionsof the present invention include:

Streptococcal antigens such as from Group A Streptococcus, or Group BStreptococcus, antigens are suitably derived from HIV-1, (such as F4antigen or fragments thereof or gag or fragments thereof such as p24,tat, nef, gp120 or gp160 or fragments of any of these), human herpesviruses, such as gD or derivatives thereof or Immediate Early proteinsuch as ICP27 from HSV1 or HSV2, cytomegalovirus ((esp Human)(such as gBor derivatives thereof), Rotavirus (including live-attenuated viruses),Epstein Barr virus (such as gp350 or derivatives thereof), VaricellaZoster Virus (such as gpI, II and IE63), or from a hepatitis virus suchas hepatitis B virus (for example Hepatitis B Surface antigen or aderivative thereof), hepatitis A virus, hepatitis C virus and hepatitisE virus, or from other viral pathogens, such as paramyxoviruses:Respiratory Syncytial virus (such as F, N and G proteins or derivativesthereof), parainfluenza virus, measles virus, mumps virus, humanpapilloma viruses (for example HPV6, 11, 16, 18,), flaviviruses (e.g.Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus) or Influenza virus (whole live orinactivated virus, split influenza virus, grown in eggs or MDCK cells,or whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10,915-920) or purified or recombinant proteins thereof, such as HA, NP,NA, or M proteins, or combinations thereof), or derived from bacterialpathogens such as Neisseria spp, including N. gonorrhea and N.meningitidis (for example capsular saccharides and conjugates thereof,transferrin-binding proteins, lactoferrin binding proteins, PiIC,adhesins); S. pyogenes (for example M proteins or fragments thereof, C5Aprotease, lipoteichoic acids), S. agalactiae, S. mutans; H. ducreyi;Moraxella spp, including M. catarrhalis, also known as Branhamellacatarrhalis (for example high and low molecular weight adhesins andinvasins); Bordetella spp, including B. pertussis (for examplepertactin, pertussis toxin or derivatives thereof, filamenteoushemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B.bronchiseptica; Mycobacterium spp., including M. tuberculosis (forexample ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof,heat-stable toxin or derivatives thereof), enterohemorragic E. coli,enteropathogenic E. coli (for example shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (for examplecholera toxin or derivatives thereof); Shigella spp, including S.sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.enterocolitica (for example a Yop protein), Y pestis, Y.pseudotuberculosis; Campylobacter spp, including C. jejuni (for exampletoxins, adhesins and invasins) and C. coli; Salmonella spp, including S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp.,including L. monocytogenes; Helicobacter spp, including H. pylori (forexample urease, catalase, vacuolating toxin); Pseudomonas spp, includingP. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani (for example tetanus toxin and derivative thereof),C. botulinum (for example botulinum toxin and derivative thereof), C.difficile (for example clostridium toxins A or B and derivativesthereof); Bacillus spp., including B. anthracis (for example botulinumtoxin and derivatives thereof); Corynebacterium spp., including C.diphtheriae (for example diphtheria toxin and derivatives thereof);Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA,DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (forexample OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC,DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp., including C. trachomatis (for example MOMP,heparin-binding proteins), C. pneumoniae (for example MOMP,heparin-binding proteins), C. psittaci; Leptospira spp., including L.interrogans; Treponema spp., including T. pallidum (for example the rareouter membrane proteins), T. denticola, T. hyodysenteriae; or derivedfrom parasites such as Plasmodium spp., including P. falciparum;Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34);Entamoeba spp., including E. histoLytica; Babesia spp., including B.microti; Trypanosoma spp., including T. cruzi; Giardia spp., includingG. lamblia; Leshmania spp., including L. major; Pneumocystis spp.,including P. carinii; Trichomonas spp., including T. vaginalis;Schisostoma spp., including S. mansoni, or derived from yeast such asCandida spp., including C. albicans; Cryptococcus spp., including C.neoformans.

Other preferred specific antigens for M. tuberculosis are for example TbRa12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO99/51748), Mtb72F and M72. Proteins for M. tuberculosis also includefusion proteins and variants thereof where at least two, preferablythree polypeptides of M. tuberculosis are fused into a larger protein.Preferred fusions include Ra12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL,Erd14-DPV-MTI-MSL-mTCC2, Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2,TbH9-DPV-MTI (WO 99/51748). A particular Ra12-Tbh9-Ra35 sequence thatmay be mentioned is defined by SEQ ID No 6 of WO2006/117240 togetherwith variants in which Ser 704 of that sequence is mutated to other thanserine, eg to Ala, and derivatives thereof incorporating an N-terminalHis tag of an appropriate length (eg SEQ ID No 2 or 4 ofWO2006/117240)”. Exemplary antigens for Chlamydia sp eg C trachomatisareselected from CT858, CT 089, CT875, MOMP, CT622, PmpD, PmpG andfragments thereof, SWIB and immunogenic fragments of any one thereof(such as PmpDpd and PmpGpd) and combinations thereof. Preferredcombinations of antigens include CT858, CT089 and CT875. Specificsequences and combinations that may be employed are described inWO2006/104890. Preferred bacterial compositions comprise antigensderived from Haemophilus spp., including H. influenzae type B (forexample PRP and conjugates thereof), non typeable H. influenzae, forexample OMP26, high molecular weight adhesins, P5, P6, protein D andlipoprotein D, and fimbrin and fimbrin derived peptides (U.S. Pat. No.5,843,464) or multiple copy varients or fusion proteins thereof.

Derivatives of Hepatitis B Surface antigen are well known in the art andinclude, inter alia, those PreS1, PreS2 S antigens set forth describedin European Patent applications EP-A-414 374; EP-A-0304 578, and EP198-474. In one preferred aspect the vaccine formulation of theinvention comprises the HIV-1 antigen, gp120, especially when expressedin CHO cells. In a further embodiment, the composition of the inventioncomprises gD2t as hereinabove defined.

The compositions may also contain an anti-tumour antigen and be usefulfor the immunotherapeutic treatment of cancers. For example, the antigenmay be a tumour rejection antigens such as those for prostrate, breast,colorectal, lung, pancreatic, renal or melanoma cancers. Exemplaryantigens include MAGE 1, 3 and MAGE 4 or other MAGE antigens such asdisclosed in WO99/40188, PRAME, BAGE, Lage (also known as NY Eos 1) SAGEand HAGE (WO 99/53061) or GAGE (Robbins and Kawakami, 1996, CurrentOpinions in Immunology 8, pps 628-636; Van den Eynde et al.,International Journal of Clinical & Laboratory Research (submitted1997); Correale et al. (1997), Journal of the National Cancer Institute89, p293. Indeed these antigens are expressed in a wide range of tumourtypes such as melanoma, lung carcinoma, sarcoma and bladder carcinoma.MAGE antigens for use in the present invention may be expressed as afusion protein with an expression enhancer or an Immunological fusionpartner. In particular, the Mage protein may be fused to Protein D fromHeamophilus infuenzae B or a lipidated derivative thereof. Inparticular, the fusion partner may comprise the first 1/3 of Protein D.Such constructs are disclosed in Wo99/40188.

Other tumour-specific antigens include, but are not restricted to KSA(GA733) tumour-specific gangliosides such as GM 2, and GM3 or conjugatesthereof to carrier proteins; or said antigen may be a self peptidehormone such as whole length Gonadotrophin hormone releasing hormone(GnRH, WO 95/20600), a short 10 amino acid long peptide, useful in thetreatment of many cancers, or in immunocastration.

In a preferred embodiment prostate antigens are utilised, such asProstate specific antigen (PSA), PAP, PSCA (PNAS 95(4) 1735-1740 1998),PSMA or antigen known as Prostase. Prostase is a prostate-specificserine protease (trypsin-like), 254 amino acid-long, with a conservedserine protease catalytic triad H-D-S and a amino-terminalpre-propeptide sequence, indicating a potential secretory function (P.Nelson, Lu Gan, C. Ferguson, P. Moss, R. Gelinas, L. Hood & K. Wand,“Molecular cloning and characterisation of prostase, anandrogen-regulated serine protease with prostate restricted expression,In Proc. Natl. Acad. Sci. USA (1999) 96, 3114-3119). A putativeglycosylation site has been described. The predicted structure is verysimilar to other known serine proteases, showing that the maturepolypeptide folds into a single domain. The mature protein is 224 aminoacids-long, with one A2 epitope shown to be naturally processed.

Prostase nucleotide sequence and deduced polypeptide sequence andhomologs are disclosed in Ferguson, et al. (Proc. Natl. Acad. Sci. USA1999, 96, 3114-3119) and in International Patent Applications No. WO98/12302 (and also the corresponding granted patent U.S. Pat. No.5,955,306), WO 98/20117 (and also the corresponding granted U.S. Pat.No. 5,840,871 and U.S. Pat. No. 5,786,148) (prostate-specifickallikrein) and WO 00/04149 (P703P). The present invention providescompositions comprising prostase protein fusions based on prostaseprotein and fragments and homologues thereof (“derivatives”). Suchderivatives are suitable for use in therapeutic vaccine formulationswhich are suitable for the treatment of a prostate tumours. Typicallythe fragment will contain at least 20, preferably 50, more preferably100 contiguous amino acids as disclosed in the above referenced patentand patent applications.

A further preferred prostate antigen is known as P501S, sequence ID no113 of Wo98/37814. Immunogenic fragments and portions thereof comprisingat least 20, preferably 50, more preferably 100 contiguous amino acidsas disclosed in the above referenced patent application. See for examplePS108 (WO 98/50567).

Other prostate specific antigens are known from Wo98/37418, andWO/004149. Another is STEAP PNAS 96 14523 14528 7-12 1999.

Other tumour associated antigens useful in the context of the presentinvention include: Plu-1 J Biol. Chem 274 (22) 15633-15645, 1999,HASH-1, HasH-2, Cripto (Salomon et al Bioessays 199, 21 61-70,U.S. Pat.No. 5,654,140) Criptin U.S. Pat. No. 5,981,215, . Additionally, antigensparticularly relevant for therapy of cancer also comprise tyrosinase andsurvivin. Mucin dervied peptides such as Muc1 see for example U.S. Pat.No. 5,744,144 U.S. Pat. No. 5,827,666 WO 8805054, U.S. Pat. No.4,963,484. Specifically contemplated are Muc 1 derived peptides thatcomprise at least one repeat unit of the Muc 1 peptide, preferably atleast two such repeats and which is recognised by the SM3 antibody (U.S.Pat. No. 6,054,438). Other mucin derived peptides include peptide fromMuc 5.

The antigen of the invention may be a breast cancer antigens such as her2/Neu, mammaglobin (U.S. Pat. No. 5,668,267) or those disclosed in WO/0052165, WO99/33869, WO99/19479, WO 98/45328. Her 2 neu antigens aredisclosed inter alia, in U.S. Pat. No. 5,801,005. Preferably the Her 2neu comprises the entire extracellular domain (comprising approximatelyamino acid 1-645) or fragments thereof and at least an immunogenicportion of or the entire intracellular domain approximately the Cterminal 580 amino acids. In particular, the intracellular portionshould comprise the phosphorylation domain or fragments thereof. Suchconstructs are disclosed in WO00/44899. A particularly preferredconstruct is known as ECD PD a second is known as ECD PD SeeWo/00/44899. The her 2 neu as used herein can be derived from rat, mouseor human.

The compositions may contain antigens associated with tumour-supportmechanisms (e.g. angiogenesis, tumour invasion) for example tie 2, VEGF.

It is foreseen that compositions of the present invention may useantigens derived from Borrelia sp. For example, antigens may includenucleic acid, pathogen derived antigen or antigenic preparations,recombinantly produced protein or peptides, and chimeric fusionproteins. In particular the antigen is OspA. The OspA may be a fullmature protein in a lipidated form virtue of the host cell (E.Coli)termed (Lipo-OspA) or a non-lipidated derivative. Such non-lipidatedderivatives include the non-lipidated NS1-OspA fusion protein which hasthe first 81 N-terminal amino acids of the non-structural protein (NS1)of the influenza virus, and the complete OspA protein, and another,MDP-OspA is a non-lipidated form of OspA carrying 3 additionalN-terminal amino acids.

Compositions of the present invention may be used for the prophylaxis ortherapy of allergy. Such vaccines would comprise allergen specific (forexample Der p1) and allergen non-specific antigens (for example peptidesderived from human IgE, including but not restricted to the stanworthdecapeptide (EP 0 477 231 B1)).

Compositions of the present invention may also be used for theprophylaxis or therapy of chronic disorders others than allergy, canceror infectious diseases. Such chronic disorders are diseases such asatherosclerosis, and Alzheimer.

The compositions of the present invention are particularly suited forthe immunotherapeutic treatment of diseases, such as chronic conditionsand cancers, but also for the therapy of persistent infections.Accordingly the compositions of the present invention are particularlysuitable for the immunotherapy of infectious diseases, such asTuberculosis (TB), AIDS and Hepatitis B (HepB) virus infections.

Also, in the context of AIDS, there is provided a method of treatment ofan individual susceptible to or suffering from AIDS. The methodcomprising the administration of a vaccine of the present invention tothe individual, thereby reducing the amount of CD4+ T-cell declinecaused by subsequent HIV infection, or slowing or halting the CD4+T-cell decline in an individual already infected with HIV.

Other antigens include bacterial (preferably capsular) saccharides otherthan (or in addition to) those pneumococcal antigens described above.Polysaccharide antigens are conveniently stored in liquid bulk adsorbedonto aluminium phosphate—it is therefore straightforward to generatevaccine compositions of the invention by admixing said liquid bulk withthe adjuvant of the invention extemporaneously. Preferably the otherbacterial saccharides are selected from a group consisting of: N.meningitidis serogroup A capsular saccharide (MenA), N. meningitidisserogroup C capsular saccharide (MenC), N. meningitidis serogroup Ycapsular saccharide (MenY), N. meningitidis serogroup W-135 capsularsaccharide (MenW), Group B Streptococcus group I capsular saccharide,Group B Streptococcus group II capsular saccharide, Group BStreptococcus group III capsular saccharide, Group B Streptococcus groupIV capsular saccharide, Group B Streptococcus group V capsularsaccharide, Staphylococcus aureus type 5 capsular saccharide,Staphylococcus aureus type 8 capsular saccharide, Vi saccharide fromSalmonella typhi, N. meningitidis LPS, M. catarrhalis LPS, and H.influenzae LPS. By LPS it is meant either native lipo-polysaccharide (orlipo-oligosaccharide), or lipo-polysaccharide where the lipid A portionhas been detoxified by any of a number of known methods (see for exampleWO 97/18837 or WO 98/33923), or any molecule comprising theO-polysaccharide derived from said LPS. By N. meningitidis LPS it ismeant one or more of the 12 known immunotypes (L1, L2, L3, L4, L5, L6,L7, L8, L9, L10, L11 or L12).

Particularly preferred combinations are compositions comprising: 1)conjugated Hib, conjugated MenA and conjugated MenC; 2) conjugated Hib,conjugated MenY and conjugated MenC; 3) conjugated Hib and conjugatedMenC; and 4) conjugated MenA, conjugated MenC, conjugated MenY andconjugated MenW-135. The amount of PS in each of the above conjugatesmay be 5 or 10 g each per 0.5 mL human dose. Preferably Hib, MenA, MenC,MenW-135 and MenY are TT conjugates.

A problem associated with the polysaccharide approach to vaccination, isthe fact that polysaccharides per se are poor immunogens. To overcomethis, saccharides of the invention may be conjugated to proteincarriers, which provide bystander T-cell help. It is preferred,therefore, that the saccharides utilised in the invention are linked tosuch a protein carrier. Examples of such carriers which are currentlycommonly used for the production of saccharide immunogens include theDiphtheria and Tetanus toxoids (DT, DT CRM197 and TT respectively),Keyhole Limpet Haemocyanin (KLH), protein D from Haemophilus influenzae(EP 594610-B), OMPC from N. meningitidis, and the purified proteinderivative of Tuberculin (PPD).

The saccharide may be linked to the carrier protein by any known method(for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al.,U.S. Pat. No. 4,474,757). Preferably, CDAP conjugation is carried out(WO 95/08348).

Preferably the protein:saccharide (weight:weight) ratio of theconjugates is 0.3:1 to 1:1, more preferably 0.6:1 to 0.8:1, and mostpreferably about 0.7:1.

Combinations of antigens which provide protection against pneumococcusand a different pathogen are included in the present invention. ManyPaediatric vaccines are now given as a combination vaccine so as toreduce the number of injections a child has to receive. Thus forPaediatric vaccines other antigens from other pathogens may beformulated with the pneumococcal vaccines of the invention. For examplethe vaccines of the invention can be formulated with (or administeredseparately but at the same time) the well known ‘trivalent’ combinationvaccine comprising Diphtheria toxoid (DT), tetanus toxoid (TT), andpertussis components [typically detoxified Pertussis toxoid (PT) andfilamentous haemagglutinin (FHA) with optional pertactin (PRN) and/oragglutinin 1+2], for example the marketed vaccine INFANRIX-DTPa™(SmithKlineBeecham Biologicals) which contains DT, TT, PT, FHA and PRNantigens, or with a whole cell pertussis component for example asmarketed by SmithKlineBeecham Biologicals s.a., as Tritanrix™. Thecombined vaccine may also comprise other antigen, such as Hepatitis Bsurface antigen (HBsAg), Polio virus antigens (for instance inactivatedtrivalent polio virus—IPV), Moraxella catarrhalis outer membraneproteins, non-typeable Haemophilus influenzae proteins, N.meningitidis Bouter membrane proteins.

Examples of preferred Moraxella catarrhalis protein antigens which canbe included in a combination vaccine (especially for the prevention ofotitis media) are: OMP106 [WO 97/41731 (Antex) & WO 96/34960 (PMC)];OMP21; LbpA &/or LbpB [WO 98/55606 (PMC)]; TbpA &/or TbpB [WO 97/13785 &WO 97/32980 (PMC)]; CopB [Helminen M E, et al. (1993) Infect. Immun.61:2003-2010]; UspA1 and/or UspA2 [WO 93/03761 (University of Texas)];OmpCD; HasR (PCT/EP99/03824); PiIQ (PCT/EP99/03823); OMP85(PCT/EP00/01468); lipo06 (GB 9917977.2); lipo10 (GB 9918208.1); lipo11(GB 9918302.2); lipo18 (GB 9918038.2); P6 (PCT/EP99/03038); D15(PCT/EP99/03822); OmpIA1 (PCT/EP99/06781); Hly3 (PCT/EP99/03257); andOmpE. Examples of non-typeable Haemophilus influenzae antigens which canbe included in a combination vaccine (especially for the prevention ofotitis media) include: Fimbrin protein [(U.S. Pat. No. 5,766,608—OhioState Research Foundation)] and fusions comprising peptides therefrom[eg LB1(f) peptide fusions; U.S. Pat. No. 5,843,464 (OSU) or WO99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673 (StateUniversity of New York)]; TbpA and/or TbpB; Hia; Hsf; Hin47; Hif; Hmw1;Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); protein D (EP 594610); P2; andP5 (WO 94/26304).

Other combinations contemplated are the pneumococcal saccharide &protein of the invention in combination with viral antigens, forexample, from influenza (attenuated, split, or subunit [e.g., surfaceglycoproteins neuraminidase (NA) and haemagglutinin (HA). See, e.g.,Chaloupka I. et al, Eur. Journal Clin. Microbiol. Infect. Dis. 1996,15:121-127], RSV (e.g., F and G antigens or F/G fusions, see, eg,Schmidt A. C. et al, J Virol, May 2001, p4594-4603), PIV3 (e.g., HN andF proteins, see Schmidt et al. supra), Varicella (e.g., attenuated,glycoproteins I-V, etc.), and any (or all) component(s) of MMR (measles,mumps, rubella). A preferred Paediatric combination vaccine contemplatedby the present invention for global treatment or prevention of otitismedia comprises: one or more Streptococcus pneumoniae saccharideantigen(s) (preferably conjugated to protein D), one or morepneumococcal proteins (preferably those described above), and one ormore surface-exposed antigen from Moraxella catarrhalis and/ornon-typeable Haemophilus influenzae. Protein D can advantageously beused as a protein carrier for the pneumococcal saccharides (as mentionedabove), and because it is in itself an immunogen capable of producingB-cell mediated protection against non-typeable H. influenzae (ntHi).The Moraxella catarrhalis or non-typeable Haemophilus influenzaeantigens can be included in the vaccine in a sub-unit form, or may beadded as antigens present on the surface of outer membrane vesicles(blebs) made from the bacteria.

Immunogenic Properties of the Immunogenic Composition Used for theVaccination of the Present Invention

In the present invention the immunogenic composition is preferablycapable of inducing an improved CD4 T-cell immune response against atleast one of the component antigen(s) or antigenic composition comparedto the CD4 T-cell immune response obtained with the correspondingcomposition which in un-adjuvanted, i.e. does not contain any exogeneousadjuvant (herein also referred to as ‘plain composition’). In a specificembodiment, where the immunogenic composition is an influenzacomposition and where the influenza vaccine preparation is from severalinfluenza strains, one of which being a pandemic starin, said improvedCD4 T-cell immune response is against the pandemic influenza strain.

By “improved CD4 T-cell immune response” is meant that a higher CD4response is obtained in a mammal after administration of the adjuvantedimmunogenic composition than that obtained after administration of thesame composition without adjuvant. For example, a higher CD4 T-cellresponse is obtained in a human patient upon administration of animmunogenic composition comprising an influenza virus or antigenicpreparation thereof together with adjuvant according to the invention,compared to the response induced after administration of an immunogeniccomposition comprising an influenza virus or antigenic preparationthereof which is un-adjuvanted. Such formulation will advantageously beused to induce anti-influenza CD4-T cell response capable of detectionof influenza epitopes presented by MHC class II molecules.

In particular but not exclusively, said ‘improved CD4 T-cell immuneresponse’ is obtained in an immunologically unprimed patient, i.e. apatient who is seronegative to said influenza virus or antigen. Thisseronegativity may be the result of said patient having never faced suchvirus or antigen (so-called ‘naive’ patient) or, alternatively, havingfailed to respond to said antigen once encountered. In a specific aspectsaid CD4 T-cell immune response is obtained in an immunocompromisedsubject such as an elderly, typically 65 years of age or above, or anadult younger than 65 years of age with a high risk medical condition(‘high risk’ adult), or a child under the age of two.

The improved CD4 T-cell immune response may be assessed by measuring thenumber of cells producing any of the following cytokines:

-   -   cells producing at least two different cytokines (CD40L, IL-2,        IFNγ, TNFα)    -   cells producing at least CD40L and another cytokine (IL-2, TNFα,        IFNγ)    -   cells producing at least IL-2 and another cytokine (CD40L, TNFα,        IFNγ)    -   cells producing at least IFNγ and another cytokine (IL-2, TNFα,        CD40L)    -   cells producing at least TNFα and another cytokine (IL-2, CD40L,        IFNγ)

There will be improved CD4 T-cell immune response when cells producingany of the above cytokines will be in a higher amount followingadministration of the adjuvanted composition compared to theadministration of the un-adjuvanted composition. Typically at least one,preferably two of the five conditions mentioned herein above will befulfilled. In a particular embodiment, the cells producing all fourcytokines will be present at a higher amount in the adjuvanted groupcompared to the un-adjuvanted group.

The improved CD4 T-cell immune response conferred by an adjuvantedinfluenza composition of the present invention may be ideally obtainedafter one single administration. The single dose approach will beextremely relevant for example in a rapidly evolving outbreak situation.In certain circumstances, especially for the elderly population, or inthe case of young children (below 9 years of age) who are vaccinated forthe first time against influenza, or in the case of a pandemics, it maybe beneficial to administer two doses of the same composition for thatseason. The second dose of said same composition (still considered as‘composition for first vaccination’) may be administered during theon-going primary immune response and is adequately spaced. Typically thesecond dose of the composition is given a few weeks, or about one month,e.g. 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after the firstdose, to help prime the immune system in unresponsive or poorlyresponsive individuals.

In another embodiment, the administration of said immunogeniccomposition induces an improved B-memory cell response in patientsadministered with the adjuvanted immunogenic composition compared to theB-memory cell response induced in individuals immunized with theun-adjuvanted composition. An improved B-memory cell response isintended to mean an increased frequency of peripheral blood Blymphocytes capable of differentiation into antibody-secreting plasmacells upon antigen encounter as measured by stimulation of in-vitrodifferentiation.

In another embodiment, the administration of said immunogeniccomposition induces an improved humoral response in patientsadministered with the adjuvanted immunogenic composition compared to thehumoral response induced in individuals immunized with the un-adjuvantedcomposition. Said humoral immune response may be measured according toany of the procedure detailed in Example 1, and especially in sectionsI.1 (I.1.1), I.2 (I.2.1) and I.3 (I.3.5.2). When the immunogeniccomposition is an influenza composition specifically said humoralresponse is obtained against homologous and heterologous strains. Inparticular, said heterologous humoral immune response means a humoralresponse between influenza strains, and is termed ‘cross-reactive’humoral immune response. Said ‘cross-reactive’ humoral immune responseinvolves the induction of response against an influenza strain which isa variant (a drift) of the influenza strain used for vaccination. Anexample of such a response is illustrated in Example III.3.1 and in FIG.2.

In a specific embodiment, the administration of said adjuvantedimmunogenic composition induces at least two of the following responses:(i) an improved CD4 T-cell immune response, (ii) an improved B-memorycell response, (iii) an improved humoral response, against at least oneof the component antigen(s) or antigenic composition compared to eitherimmune response obtained with the corresponding composition which inun-adjuvanted, i.e. does not contain any exogeneous adjuvant (hereinalso referred to as ‘plain composition’).

In a still further specific embodiment, the vaccination with thecomposition for the first vaccination, adjuvanted, has no measurableimpact on the CD8 response.

It is a specific embodiment of the invention that the compositioncomprising an influenza virus or antigenic preparation thereofformulated with saponin adjuvant presented in the form of a liposome, inparticular QS21 saponin in its quenched form with cholesterol, iseffective in promoting T cell responses in an immuno-compromised humanpopulation. In one embodiment, said adjuvant further comprises 3D-MPL.In particular, the administration of a single dose of the immunogeniccomposition for first vaccination as described in the invention, iscapable of providing better sero-protection, as assessed by thecorrelates of protection for influenza vaccines, following revaccinationagainst influenza in a human elderly population, than does thevaccination with an un-adjuvanted influenza vaccine. The claimedadjuvanted formulation has also been able to induce an improved CD4T-cell immune response against influenza virus compared to that obtainedwith the un-adjuvanted formulation. This finding can be associated withan increased responsiveness upon vaccination or infection vis-à-visinfluenza antigenic exposure. Furthermore, this may also be associatedwith a cross-responsiveness, i.e. a higher ability to respond againstvariant influenza strains. This improved response may be especiallybeneficial in an immuno-compromised human population such as the elderlypopulation (65 years of age and above) and in particular the high riskelderly population. This may result in reducing the overall morbidityand mortality rate and preventing emergency admissions to hospital forpneumonia and other influenza-like illness. This may also be of benefitto the infant population (below 5 years, preferably below 2 years ofage). Furthermore it may allow inducing a CD4 T cell response which ismore persistent in time, e.g. still present one year after the firstvaccination, compared to the response induced with the un-adjuvantedformulation.

In a specific aspect, the CD4 T-cell immune response, such as theimproved CD4 T-cell immune response obtained in an unprimed subject,involves the induction of a cross-reactive CD4 T helper response. Inparticular, the amount of cross-reactive CD4 T cells is increased. By‘cross-reactive’ CD4 response is meant CD4 T-cell targeting sharedepitopes between influenza strains.

Usually, available influenza vaccines are effective only againstinfecting strains of influenza virus that have haemagglutinin of similarantigenic characteristics. When the infecting (circulating) influenzavirus has undergone minor changes (such as a point mutation or anaccumulation of point mutations resulting in amino acid changes in thefor example) in the surface glycoproteins in particular haemagglutinin(antigenic drift variant virus strain) the vaccine may still providesome protection, although it may only provide limited protection as thenewly created variants may escape immunity induced by prior influenzainfection or vaccination. Antigenic drift is responsible for annualepidemics that occur during interpandemic periods (Wiley & Skehel, 1987,Ann. Rev. Biochem. 56, 365-394). The induction of cross-reactive CD4 Tcells provides an additional advantage to the composition of theinvention, in that it may provide also cross-protection, in other wordsprotection against heterologous infections, i.e. infections caused by acirculating influenza strain which is a variant (e.g. a drift) of theinfluenza strain contained in the immunogenic composition. This may beadvantageous when the circulating strain is difficult to propagate ineggs or to produce in cell culture, rendering the use of a driftedstrain a working alternative. This may also be advantageous when thesubject received a first and a second vaccination several months or ayear apart, and the influenza strain in the immunogenic composition usedfor a second immunization is a drift variant strain of the strain usedin the composition used for the first vaccination.

The adjuvanted influenza immunogenic composition as herein defined hastherefore a higher ability to induce sero-protection and cross-reactiveCD4 T cells in vaccinated elderly subjects. This characteristic may beassociated with a higher ability to respond against a variant strain ofthe strain present in the immunogenic composition. This may prove to bean important advantage in a pandemic situation. For example amultivalent influenza immunogenic composition comprising any or severalof H5, a H2, a H9, H7 or H6 strain(s) may provide a higher ability torespond against a pandemic variant, i.e. a drift strain of said pandemicstrain(s), either upon subsequent vaccination with or upon infection bysaid drift strain.

Detection of Cross-Reactive CD4 T-Cells Following Vaccination withInfluenza Vaccine

CD4 T-cells that are able to recognize both homologous and driftedInfluenza strains have been named in the present document“cross-reactive”. The adjuvanted influenza compositions as describedherein have been capable to show heterosubtypic cross-reactivity sincethere is observable cross-reactivity against drifted Influenza strains.As said above, the ability of a pandemic vaccine formulation to beeffective against drift pandemic strains may prove to be an importantcharacteristic in the case of pandemics.

Consistently with the above observations, CD4 T-cell epitopes shared bydifferent Influenza strains have been identified in human (Gelder C etal. 1998, Int Immunol. 10(2):211-22; Gelder C M et al. 1996 J Virol.70(7):4787-90; Gelder C M et al. 1995 J Virol. 1995 69(12):7497-506).

In a specific embodiment, the adjuvanted composition may offer theadditional benefit of providing better protection against circulatingstrains which have undergone a major change (such as gene recombinationfor example, between two different species) in the haemagglutinin(antigenic shift) against which currently available vaccines have noefficacy.

Revaccination and Composition Used for Revaccination (BoostingComposition)

In one embodiment, the invention provides for the use of an influenzavirus or antigenic preparation thereof in the manufacture of animmunogenic composition for revaccination of humans previouslyvaccinated with an immunogenic composition as claimed herein.

In one aspect of the present invention, there is provided the use of aninfluenza virus or antigenic preparation thereof, from a first pandemicinfluenza strain, in the manufacture of an adjuvanted immunogeniccomposition as herein defined for protection against influenzainfections caused by a influenza strain which is a variant of said firstinfluenza strain.

In another aspect, the invention provides for the use of an influenzavirus or antigenic preparation thereof in the manufacture of aninfluenza immunogenic composition for revaccination of humans previouslyvaccinated with an adjuvanted influenza composition as claimed herein orwith an adjuvanted influenza composition comprising a variant influenzastrain, the adjuvant being as defined herein.

In another aspect the present invention provides for a method forvaccinating a human population or individual against one influenza virusstrain followed by revaccination of said human or population against avariant influenza virus strain, said method comprising administering tosaid human (i) a first composition comprising an influenza virus orantigenic preparation thereof from a first influenza virus strain and anadjuvant as herein defined, and (ii) a second immunogenic compositioncomprising a influenza virus strain variant of said first influenzavirus strain. In a specific embodiment said first strain is associatedwith a pandemic outbreak or has the potential to be associated with apandemic outbreak. In another specific embodiment said variant strain isassociated with a pandemic outbreak or has the potential to beassociated with a pandemic outbreak. In particular, the re-vaccinationis made with an influenza composition comprising at least one strainwhich is a circulating pandemic strain. Both the priming composition andthe boosting composition can be multivalent, i.e. can contain at leasttwo influenza virus strains. When the composition(s) is (are)multivalent, at least one strain is associated with a pandemic outbreakor has the potential to be associated with a pandemic outbreak.

Typically revaccination is made at least 6 months after the firstvaccination(s), preferably 8 to 14 months after, more preferably ataround 10 to 12 months after.

The immunogenic composition for revaccination (the boosting composition)may contain any type of antigen preparation, either inactivated or liveattenuated. It may contain the same type of antigen preparation e.g. asplit influenza virus or antigenic preparation thereof, a whole virion,or a purified HA and NA (sub-unit) vaccine, as the immunogeniccomposition used for the first vaccination. Alternatively the boostingcomposition may contain another type of influenza antigen than that usedfor the first vaccination. Preferably a split virus is used. Theboosting composition may be adjuvanted or un-adjuvanted. Theun-adjuvanted boosting composition may be Fluarix™/α-Rix®/Influsplit®given intramuscularly. The formulation contains three inactivated splitvirion antigens prepared from the WHO recommended strains of theappropriate influenza season.

The boosting composition may be adjuvanted or un-adjuvanted. In aspecific embodiment, the boosting composition comprises a saponinadjuvant which is as defined herein.

In a specific embodiment, the immunogenic composition for revaccination(also called herein below the ‘boosting composition’) contains aninfluenza virus or antigenic preparation thereof which shares common CD4T-cell epitopes with the influenza virus or antigenic preparationthereof used for the first vaccination. A common CD4 T cell epitope isintended to mean peptides/sequences/epitopes from different antigenswhich can be recognised by the same CD4 cell (see examples of describedepitopes in: Gelder C et al. 1998, Int Immunol. 10(2):211-22; Gelder C Met al. 1996 J Virol. 70(7):4787-90; Gelder C M et al. 1995 J Virol. 199569(12):7497-506).

In an embodiment according to the invention, the boosting composition isa monovalent influenza composition comprising an influenza strain whichis associated with a pandemic outbreak or has the potential to beassociated with a pandemic outbreak. In particular said strain in theboosting composition is a circulating pandemic strain. Suitable strainsare, but not limited to: H5N1, H9N2, H7N7, and H2N2. Said strain may bethe same as that, or one of those, present in the composition used forthe first vaccination. In an alternative embodiment said strain may be avariant strain, i.e. a drift strain, of the strain present in thecomposition used for the first vaccination.

In another specific embodiment, the boosting composition is amultivalent influenza vaccine. In particular, when the boostingcomposition is a multivalent vaccine such as a bivalent, trivalent orquadrivalent vaccine, at least one strain is associated with a pandemicoutbreak or has the potential to be associated with a pandemic outbreak.In a specific embodiment, two or more strains in the boostingcomposition are pandemic strains. In another specific embodiment, the atleast one pandemic strain in the boosting composition is of the sametype as that, or one of those, present in the composition used for thefirst vaccination. In an alternative embodiment the at least one strainmay be a variant strain, i.e. a drift strain, of the at least onepandemic strain present in the composition used for the firstvaccination. In particular the at least one strain in the boostingcomposition is a circulating pandemic strain. The boosting compositionmay be adjuvanted or not.

Typically a boosting composition, where used, is given at the nextinfluenza season, e.g. approximately one year after the firstimmunogenic composition. The boosting composition may also be givenevery subsequent year (third, fourth, fifth vaccination and so forth).The boosting composition may be the same as the composition used for thefirst vaccination. Suitably, the boosting composition contains aninfluenza virus or antigenic preparation thereof which is a variantstrain of the influenza virus used for the first vaccination. Inparticular, the influenza viral strains or antigenic preparation thereofare selected according to the reference material distributed by theWorld Health Organisation such that they are adapted to the influenzastrain which is circulating on the year of the revaccination.

The influenza antigen or antigenic composition used in revaccinationpreferably comprises an adjuvant, suitably as described above. Theadjuvant may be a saponin presented in the form of a liposome, as hereinabove described, which is preferred, optionally containing an additionaladjuvant such as 3D-MPL.

In one aspect revaccination induces any, preferably two or all, of thefollowing: (i) an improved CD4 response against the influenza virus orantigenic preparation thereof, or (ii) an improved B cell memoryresponse or (iii) an improved humoral response, compared to theequivalent response induced after a first vaccination with theun-adjuvanted influenza virus or antigenic preparation thereof.Preferably the immunological response(s) induced after revaccinationwith the adjuvanted influenza virus or antigenic preparation thereof asherein defined, is (are) higher than the corresponding response inducedafter the revaccination with the un-adjuvanted composition. Preferablythe immunological responses induced after revaccination with anun-adjuvanted, preferably split, influenza virus are higher in thepopulation first vaccinated with the adjuvanted, preferably split,influenza composition than the corresponding response in the populationfirst vaccinated with the un-adjuvanted, preferably split, influenzacomposition.

In a specific embodiment, the revaccination of the subjects with aboosting composition comprising an influenza virus and a saponinadjuvant in the form of a liposome, as defined herein above, showshigher antibody titers than the corresponding values in the group ofpeople first vaccinated with the un-adjuvanted composition and boostedwith the un-adjuvanted composition. The effect of the adjuvant inenhancing the antibody response to revaccination is especially ofimportance in the elderly population which is known to have a lowresponse to vaccination or infection by influenza virus. The adjuvantedcomposition-associated benefit was also marked in terms of improving theCD4 T-cell response following revaccination.

Specifically, the adjuvanted composition of the invention is capable ofinducing a better cross-responsiveness against drifted strain (theinfluenza strain from the next influenza season) compared to theprotection conferred by the control vaccine. Said cross-responsivenesshas shown a higher persistence compared to that obtained with theun-adjuvanted formulation. The effect of the adjuvant in enhancing thecross-responsiveness against drifted strain is of important in apandemic situation.

In a further embodiment the invention relates to a vaccination regime inwhich the first vaccination is made with an influenza composition,preferably a split influenza composition, containing at least oneinfluenza strain that could potentially cause a pandemic outbreak andthe revaccination is made with a circulating strain, either a pandemicstrain or a classical strain.

CD4 Epitope in HA

This antigenic drift mainly resides in epitope regions of the viralsurface proteins haemagglutinin (HA) and neuraminidase (NA). It is knownthat any difference in CD4 and B cell epitopes between differentinfluenza strains, being used by the virus to evade the adaptiveresponse of the host immune system, will play a major role in influenzavaccination and is.

CD4 T-cell epitopes shared by different Influenza strains have beenidentified in human (see for example: Gelder C et al. 1998, Int Immunol.10(2):211-22; Gelder C M et al. 1996 J Virol. 70(7):4787-90; and GelderC M et al. 1995 J Virol. 1995 69(12):7497-506).

In a specific embodiment, the revaccination is made by using a boostingcomposition which contains an influenza virus or antigenic preparationthereof which shares common CD4 T-cell epitopes with the influenza virusantigen or antigenic preparation thereof used for the first vaccination.The invention thus relates to the use of the immunogenic compositioncomprising a pandemic influenza virus or antigenic preparation thereofand a saponoin in the form of a liposome, in particular QS21 in itsdetoxified form with cholesterol optionally with 3D-MPL, in themanufacture of a first vaccination-component of a multi-dose vaccine,the multi-dose vaccine further comprising, as a boosting dose, aninfluenza virus or antigenic preparation thereof which shares common CD4T-cell epitopes with the pandemic influenza virus antigen or virusantigenic preparation thereof of the dose given at the firstvaccination.

Vaccination Means

The immunogenic compositions of the invention may be administered by anysuitable delivery route, such as intradermal, mucosal e.g. intranasal,oral, intramuscular or subcutaneous. Other delivery routes are wellknown in the art.

The intramuscular delivery route is preferred for the adjuvantedimmunogenic composition.

Intradermal delivery is another suitable route. Any suitable device maybe used for intradermal delivery, for example short needle devices suchas those described in U.S. Pat. No. 4,886,499, U.S. Pat. No. 5,190,521,U.S. Pat. No. 5,328,483, U.S. Pat. No. 5,527,288, U.S. Pat. No.4,270,537, U.S. Pat. No. 5,015,235, U.S. Pat. No. 5,141,496, U.S. Pat.No. 5,417,662. Intradermal vaccines may also be administered by deviceswhich limit the effective penetration length of a needle into the skin,such as those described in WO99/34850 and EP1092444, incorporated hereinby reference, and functional equivalents thereof. Also suitable are jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector or via a needle which pierces the stratum corneumand produces a jet which reaches the dermis. Jet injection devices aredescribed for example in U.S. Pat. No. 5,480,381, U.S. Pat. No.5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat. No. 5,993,412, U.S. Pat.No. 5,649,912, U.S. Pat. No. 5,569,189, U.S. Pat. No. 5,704,911, U.S.Pat. No. 5,383,851, U.S. Pat. No. 5,893,397, U.S. Pat. No. 5,466,220,U.S. Pat. No. 5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No.5,503,627, U.S. Pat. No. 5,064,413, U.S. Pat. No. 5,520,639, U.S. Pat.No. 4,596,556 U.S. Pat. No. 4,790,824, U.S. Pat. No. 4,941,880, U.S.Pat. No. 4,940,460, WO 97/37705 and WO 97/13537. Also suitable areballistic powder/particle delivery devices which use compressed gas toaccelerate vaccine in powder form through the outer layers of the skinto the dermis. Additionally, conventional syringes may be used in theclassical mantoux method of intradermal administration.

Another suitable administration route is the subcutaneous route. Anysuitable device may be used for subcutaneous delivery, for exampleclassical needle. Preferably, a needle-free jet injector service isused, such as that published in WO 01/05453, WO 01/05452, WO 01/05451,WO 01/32243, WO 01/41840, WO 01/41839, WO 01/47585, WO 01/56637, WO01/58512, WO 01/64269, WO 01/78810, WO 01/91835, WO 01/97884, WO02/09796, WO 02/34317. More preferably said device is pre-filled withthe liquid vaccine formulation.

Alternatively the vaccine is administered intranasally. Typically, thevaccine is administered locally to the nasopharyngeal area, preferablywithout being inhaled into the lungs. It is desirable to use anintranasal delivery device which delivers the vaccine formulation to thenasopharyngeal area, without or substantially without it entering thelungs.

Preferred devices for intranasal administration of the vaccinesaccording to the invention are spray devices. Suitable commerciallyavailable nasal spray devices include Accuspray™ (Becton Dickinson).Nebulisers produce a very fine spray which can be easily inhaled intothe lungs and therefore does not efficiently reach the nasal mucosa.Nebulisers are therefore not preferred.

Preferred spray devices for intranasal use are devices for which theperformance of the device is not dependent upon the pressure applied bythe user. These devices are known as pressure threshold devices. Liquidis released from the nozzle only when a threshold pressure is applied.These devices make it easier to achieve a spray with a regular dropletsize. Pressure threshold devices suitable for use with the presentinvention are known in the art and are described for example in WO91/13281 and EP 311 863 B and EP 516 636, incorporated herein byreference. Such devices are commercially available from Pfeiffer GmbHand are also described in Bommer, R. Pharmaceutical Technology Europe,September 1999.

Preferred intranasal devices produce droplets (measured using water asthe liquid) in the range 1 to 200 μm, preferably 10 to 120 μm. Below 10μm there is a risk of inhalation, therefore it is desirable to have nomore than about 5% of droplets below 10 μm. Droplets above 120 μm do notspread as well as smaller droplets, so it is desirable to have no morethan about 5% of droplets exceeding 120 μm.

Bi-dose delivery is a further preferred feature of an intranasaldelivery system for use with the vaccines according to the invention.Bi-dose devices contain two sub-doses of a single vaccine dose, onesub-dose for administration to each nostril. Generally, the twosub-doses are present in a single chamber and the construction of thedevice allows the efficient delivery of a single sub-dose at a time.Alternatively, a monodose device may be used for administering thevaccines according to the invention.

Alternatively, the epidermal or transdermal vaccination route is alsocontemplated in the present invention.

In a specific aspect of the present invention, the adjuvantedimmunogenic composition for the first administration may be givenintramuscularly, and the boosting composition, either adjuvanted or not,may be administered through a different route, for example intradermal,subcutaneous or intranasal. In another specific embodiment, animmunogenic composition comprising specifically an influenza virusantigen or antigenic preparation thereof for the first administrationmay contain a standard HA content of 15 μg per influenza strain, and theboosting influenza composition may contain a low dose of HA, i.e. below15 μg, and depending on the administration route, may be given in asmaller volume.

Populations to Vaccinate

The target population to vaccinate may be immuno-compromised human.Immuno-compromised humans generally are less well able to respond to anantigen, in particular to an influenza antigen, in comparison to healthyadults.

Preferably the target population is a population which is unprimedagainst influenza, either being naïve (such as vis à vis a pandemicstrain), or having failed to respond previously to influenza infectionor vaccination. Preferably the target population is elderly personssuitably aged 65 years and over, younger high-risk adults (i.e. between18 and 64 years of age) such as people working in health institutions,or those young adults with a risk factor such as cardiovascular andpulmonary disease, or diabetes. Another target population is allchildren 6 months of age and over, especially children 6-23 months ofage who experience a relatively high influenza-related hospitalizationrate. Preferably the target population is elderly above 65 years of age.

Vaccination Regimes, Dosing and Additional Efficacy Criteria

Suitably the immunogenic compositions according to the present inventionare a standard 0.5 ml injectable dose in most cases, and, when aninfluenza composition, contains 15 μg of haemagglutinin antigencomponent from the or each influenza strain, as measured by singleradial immunodiffusion (SRD) (J. M. Wood et al.: J. Biol. Stand. 5(1977) 237-247; J. M. Wood et al., J. Biol. Stand. 9 (1981) 317-330).Suitably the vaccine dose volume will be between 0.5 ml and 1 ml, inparticular a standard 0.5 ml, or 0.7 ml vaccine dose volume. Forinfluenza vaccines, slight adaptation of the dose volume will be maderoutinely depending on the HA concentration in the original bulk sample.

Suitably said immunogenic composition contains a low dose of HAantigen—e.g any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 μg ofHA per influenza strain.

Advantageously, a vaccine dose according to the invention, in particulara low dose vaccine, may be provided in a smaller volume than theconventional injected split flu vaccines, which are generally around0.5, 0.7 or 1 ml per dose. The low volume doses according to theinvention are preferably below 500 μl, more preferably below 300 μl andmost preferably not more than about 200 μl or less per dose.

Thus, a preferred low volume vaccine dose according to one aspect of theinvention is a dose with a low antigen dose in a low volume, e.g. about15 μg or about 7.5 μg HA or about 3.0 μg HA (per strain) in a volume ofabout 200 μl.

The influenza medicament of the invention preferably meets certaininternational criteria for vaccines.

Standards are applied internationally to measure the efficacy ofinfluenza vaccines. The European Union official criteria for aneffective vaccine against influenza are set out in the Table 1 below.Theoretically, to meet the European Union requirements, an influenzavaccine has to meet only one of the criteria in the table, for allstrains of influenza included in the vaccine. The compositions of thepresent invention suitably meet at least one such criteria.

However in practice, at least two or all three of the criteria will needto be met for all strains, particularly for a new vaccine such as a newvaccine for delivery via a different route. Under some circumstances twocriteria may be sufficient. For example, it may be acceptable for two ofthe three criteria to be met by all strains while the third criterion ismet by some but not all strains (e.g. two out of three strains). Therequirements are different for adult populations (18-60 years) andelderly populations (>60 years).

TABLE 1 18-60 years >60 years Seroconversion rate* >40% >30% Conversionfactor** >2.5 >2.0 Protection rate*** >70% >60% *Seroconversion rate isdefined as the percentage of vaccinees who have at least a 4-foldincrease in serum haemagglutinin inhibition (HI) titres aftervaccination, for each vaccine strain. **Conversion factor is defined asthe fold increase in serum HI geometric mean titres (GMTs) aftervaccination, for each vaccine strain. ***Protection rate is defined asthe percentage of vaccinees with a serum HI titre equal to or greaterthan 1:40 after vaccination (for each vaccine strain) and is normallyaccepted as indicating protection.

In a further aspect the invention provides a method of designing avaccine for diseases known to be cured or treated through a CD4+ T cellactivation, comprising

-   -   1) selecting an antigen containing CD4+ epitopes, and    -   2) combining said antigen with saponin adjuvant in the form of a        liposome as defined herein above, wherein said vaccine upon        administration in said mammal is capable of inducing an enhanced        CD4 T cell response in said mammal.

The teaching of all references in the present application, includingpatent applications and granted patents, are herein fully incorporatedby reference.

For the avoidance of doubt the terms ‘comprising’, ‘comprise’ and‘comprises’ herein is intended by the inventors to be optionallysubstitutable with the terms ‘consisting of’, ‘consist of’, and‘consists of’, respectively, in every instance.

The invention will be further described by reference to the following,non-limiting, examples:

Example I describes immunological read-out methods used in mice, ferretand human studies.

Example II describes preparation of the MPL/QS21 liposomal adjuvant

Example III describes a pre-clinical evaluation of adjuvanted andunadjuvanted influenza vaccines in ferrets.

Example IV shows a pre-clinical evaluation of adjuvanted andun-adjuvanted influenza vaccines in C57BI/6 naïve and primed mice.

Example V describes a comparison of adjuvanted influenza vaccine with3D-MPL at two different concentrations in mice.

Example VI describes a comparison of adjuvanted influenza vaccine with3D-MPL at two different concentrations in elderly humans.

Example VII describes the pre-clinical evaluation of adjuvanted HPVvaccines in mice.

Example VIII describes a pre-clinical evaluation of adjuvanted andnon-adjuvanted cytomegaolovirus immunogenic compositions.

Example IX describes the pre-clinical evaluation of an adjuvanted RTS,Svaccine composition with 3D-MPL at two different concentrations.

Example X describes the clinical evaluation of an adjuvanted RTS,Svaccine with 3D-MPL at two different concentrations.

Example I Immunological Read-Out Methods

I.1. Mice Methods

I.1.1. Hemagglutination Inhibition Test

Test Procedure

Anti-Hemagglutinin antibody titers to the three influenza virus strainswere determined using the hemagglutination inhibition test (HI). Theprinciple of the HI test is based on the ability of specificanti-Influenza antibodies to inhibit hemagglutination of chicken redblood cells (RBC) by influenza virus hemagglutinin (HA). Heatinactivated sera were previously treated by Kaolin and chicken RBC toremove non-specific inhibitors. After pretreatment, two-fold dilutionsof sera were incubated with 4 hemagglutination units of each influenzastrain. Chicken red blood cells were then added and the inhibition ofagglutination was scored. The titers were expressed as the reciprocal ofthe highest dilution of serum that completely inhibitedhemagglutination. As the first dilution of sera was 1:20, anundetectable level was scored as a titer equal to 10.

Statistical Analysis

Statistical analysis were performed on post vaccination HI titers usingUNISTAT. The protocol applied for analysis of variance can be brieflydescribed as follow:

-   -   Log transformation of data    -   Shapiro-Wilk test on each population (group) in order to verify        the normality of groups distribution    -   Cochran test in order to verify the homogenicity of variance        between the different populations (groups)    -   Two-way Analysis of variance performed on groups    -   Tukey HSD test for multiple comparisons

I.1.2. Intracellular Cytokine Staining

This technique allows a quantification of antigen specific T lymphocyteson the basis of cytokine production: effector T cells and/oreffector-memory T cells produce IFN-γ and/or central memory T cellsproduce IL-2. PBMCs are harvested at day 7 post-immunization.

Lymphoid cells are re-stimulated in vitro in the presence of secretioninhibitor (Brefeldine):

These cells are then processed by conventional immunofluorescentprocedure using fluorescent antibodies (CD4, CD8, IFN-γ and IL-2).Results are expressed as a frequency of cytokine positive cell withinCD4/CD8 T cells. Intracellular staining of cytokines of T cells wasperformed on PBMC 7 days after the second immunization. Blood wascollected from mice and pooled in heparinated medium RPMI+Add. Forblood, RPMI+Add-diluted PBL suspensions were layered onto aLympholyte-Mammal gradient according to the recommended protocol(centrifuge 20 min at 2500 rpm and R.T.). The mononuclear cells at theinterface were removed, washed 2× in RPMI+Add and PBMCs suspensions wereadjusted to 2×10⁶ cells/ml in RPMI 5% fetal calf serum.

In vitro antigen stimulation of PBMCs was carried out at a finalconcentration of 1×10⁶ cells/ml (tube FACS) with Flu trivalent split onμbeads (5 μg HA/strain) or Whole F1 (1 μgHA/strain) and then incubated 2hrs at 37° C. with the addition of anti-CD28 and anti-CD49d (1 μg/ml forboth).

The addition of both antibodies, increased proliferation and cytokineproduction by activated T and NK cells and can provide a costimulatorysignal for CTL induction.

In addition, PBMCs were also stimulated overnight with Flu trivalentsplit (30 μg HA/strain)- or Whole Fl (5 μgHA/strain)-pulsed BMDCs (1×10⁵cells/ml), which were prepared by pulsing BMDCs with Flu split (60 μg/HAstrain) or Whole Flu trivalent Fl (10 μgHA/strain) for 6 hrs at 37° C.Following the antigen restimulation step, PBMC are incubated O.N. at 37°C. in presence of Brefeldin (1 μg/ml) at 37° C. to inhibit cytokinesecretion.

IFN-γ/IL-2/CD4/CD8 staining was performed as follows: Cell suspensionswere washed, resuspended in 50 μl of PBS 1% FCS containing 2% Fcblocking reagent (1/50; 2.4G2). After 10 min incubation at 4° C., 50 μlof a mixture of anti-CD4-PE (2/50) and anti-CD8 perCp (3/50) was addedand incubated 30 min at 4° C. After a washing in PBS 1% FCS, cells werepermeabilized by resuspending in 200 μl of Cytofix-Cytoperm (Kit BD) andincubated 20 min at 4° C. Cells were then washed with Perm Wash (Kit BD)and resuspended with 50 μl of a mix of anti-IFN-γ APC (1/50)+anti-IL-2FITC (1/50) diluted in Perm Wash. After an incubation min 2 h maxovernight at 4° C., cells were washed with Perm Wash and resuspended inPBS 1% FCS+1% paraformaldéhyde. Sample analysis was performed by FACS.Live cells were gated (FSC/SSC) and acquisition was performed on ˜50,000events (lymphocytes) or 35,000 events on CD4+ T cells. The percentagesof IFN-γ+ or IL2+ were calculated on CD4+ and CD8+ gated populations.

I.2. Ferrets Methods

I.2.1. Hemagglutination Inhibition Test (HI)

Test Procedure.

Anti-Hemagglutinin antibody titers to the three influenza virus strainswere determined using the hemagglutination inhibition test (HI). Theprinciple of the HI test is based on the ability of specificanti-Influenza antibodies to inhibit hemagglutination of chicken redblood cells (RBC) by influenza virus hemagglutinin (HA). Sera were firsttreated with a 25% neuraminidase solution (RDE) and wereheat-inactivated to remove non-specific inhibitors. After pretreatment,two-fold dilutions of sera were incubated with 4 hemagglutination unitsof each influenza strain. Chicken red blood cells were then added andthe inhibition of agglutination was scored using tears for reading. Thetiters were expressed as the reciprocal of the highest dilution of serumthat completely inhibited hemagglutination. As the first dilution ofsera was 1:10, an undetectable level was scored as a titer equal to 5.

Statistical Analysis.

Statistical analysis were performed on HI titers (Day 41, beforechallenge) using UNISTAT. The protocol applied for analysis of variancecan be briefly described as followed:

-   -   Log transformation of data.    -   Shapiro-wilk test on each population (group) in order to verify        the normality of groups distribution.    -   Cochran test in order to verify the homogenicity of variance        between the different populations (groups).    -   Test for interaction of one-way ANOVA.    -   Tuckey-HSD Test for multiple comparisons.

I.2.2. Nasal Washes

The nasal washes were performed by administration of 5 ml of PBS in bothnostrils in awake animals. The inoculum was collected in a Petri dishand placed into sample containers on dry ice.

Viral Titration in Nasal Washes

All nasal samples were first sterile filtered through Spin X filters(Costar) to remove any bacterial contamination. 50 μl of serial ten-folddilutions of nasal washes were transferred to microtiter platescontaining 50 μl of medium (10 wells/dilution). 100 μl of MDCK cells(2.4×10⁵ cells/ml) were then added to each well and incubated at 35° C.for 5-7 days. After 6-7 days of incubation, the culture medium is gentlyremoved and 100 μl of a 1/20 WST-1 containing medium is added andincubated for another 18 hrs.

The intensity of the yellow formazan dye produced upon reduction ofWST-1 by viable cells is proportional to the number of viable cellspresent in the well at the end of the viral titration assay and isquantified by measuring the absorbance of each well at the appropriatewavelength (450 nanometers). The cut-off is defined as the OD average ofuninfected control cells—0.3 OD (0.3 OD correspond to +/−3 StDev of ODof uninfected control cells). A positive score is defined when OD is<cut-off and in contrast a negative score is defined when ODis >cut-off. Viral shedding titers were determined by “Reed and Muench”and expressed as Log TCID50/ml.

I.3. Assays for Assessing the Immune Response in Humans

I.3.1. Hemagglutination Inhibition Assay

The immune response was determined by measuring HI antibodies using themethod described by the WHO Collaborating Centre for influenza, Centresfor Disease Control, Atlanta, USA (1991).

Antibody titre measurements were conducted on thawed frozen serumsamples with a standardised and comprehensively validated micromethodusing 4 hemagglutination-inhibiting units (4 HIU) of the appropriateantigens and a 0.5% fowl erythrocyte suspension. Non-specific seruminhibitors were removed by heat treatment and receptor-destroyingenzyme.

The sera obtained were evaluated for HI antibody levels. Starting withan initial dilution of 1:10, a dilution series (by a factor of 2) wasprepared up to an end dilution of 1:20480. The titration end-point wastaken as the highest dilution step that showed complete inhibition(100%) of hemagglutination. All assays were performed in duplicate.

I.3.2. Neuraminidase Inhibition Assay

The assay was performed in fetuin-coated microtitre plates. A 2-folddilution series of the antiserum was prepared and mixed with astandardised amount of influenza A H3N2, H1N1 or influenza B virus. Thetest was based on the biological activity of the neuraminidase whichenzymatically releases neuraminic acid from fetuin. After cleavage ofthe terminal neuraminic acid β-D-glactose-N-acetyl-galactosamin wasunmasked. Horseradish peroxidase (HRP)-labelled peanut agglutinin fromArachis hypogaea, which binds specifically to the galactose structures,was added to the wells. The amount of bound agglutinin can be detectedand quantified in a substrate reaction with tetra-methylbenzidine (TMB)The highest antibody dilution that still inhibits the viralneuraminidase activity by at least 50% was indicated is the NI titre.

I.3.3. Neutralising Antibody Assay

Neutralising antibody measurements were conducted on thawed frozen serumsamples. Virus neutralisation by antibodies contained in the serum wasdetermined in a microneutralization assay. The sera were used withoutfurther treatment in the assay. Each serum was tested in triplicate. Astandardised amount of virus was mixed with serial dilutions of serumand incubated to allow binding of the antibodies to the virus. A cellsuspension, containing a defined amount of MDCK cells was then added tothe mixture of virus and antiserum and incubated at 33° C. After theincubation period, virus replication was visualised by hemagglutinationof chicken red blood cells. The 50% neutralisation titre of a serum wascalculated by the method of Reed and Muench.

I.3.4. Cell-Mediated Immunity was Evaluated by Cytokine Flow Cytometry(CFC)

Peripheral blood antigen-specific CD4 and CD8 T cells can berestimulated in vitro to produce IL-2, CD40L, TNF-alpha and IFN ifincubated with their corresponding antigen. Consequently,antigen-specific CD4 and CD8 T cells can be enumerated by flow cytometryfollowing conventional immunofluorescence labelling of cellularphenotype as well as intracellular cytokines production. In the presentstudy, Influenza vaccine antigen as well as peptides derived fromspecific influenza protein were used as antigen to restimulateInfluenza-specific T cells. Results were expressed as a frequency ofcytokine(s)-positive CD4 or CD8 T cell within the CD4 or CD8 T cellsub-population.

I.3.5. Statistical Methods

I.3.5.1. Primary Endpoints

-   -   Percentage, intensity and relationship to vaccination of        solicited local and general signs and symptoms during a 7 day        follow-up period (i.e. day of vaccination and 6 subsequent days)        after vaccination and overall.    -   Percentage, intensity and relationship to vaccination of        unsolicited local and general signs and symptoms during a 21 day        follow-up period (i.e. day of vaccination and 20 subsequent        days) after vaccination and overall.    -   Occurrence of serious adverse events during the entire study.

I.3.5.2. Secondary Endpoints

For the Humoral Immune Response:

Observed Variables:

-   -   At days 0 and 21: serum hemagglutination-inhibition (HI) and NI        antibody titres, tested separately against each of the three        influenza virus strains represented in the vaccine (anti-H1N1,        anti-H3N2 & anti-B-antibodies).    -   At days 0 and 21: neutralising antibody titres, tested        separately against each of the three influenza virus strains        represented in the vaccine

Derived Variables (with 95% Confidence Intervals):

-   -   Geometric mean titres (GMTs) of serum HI antibodies with 95%        confidence intervals (95% CI) pre and post-vaccination    -   Seroconversion rates* with 95% CI at day 21    -   Conversion factors** with 95% CI at day 21    -   Seroprotection rates*** with 95% CI at day 21    -   Serum NI antibody GMTs' (with 95% confidence intervals) at all        timepoints. *Seroconversion rate defined as the percentage of        vaccinees who have at least a 4-fold increase in serum HI titres        on day 21 compared to day 0, for each vaccine        strain.**Conversion factor defined as the fold increase in serum        HI GMTs on day 21 compared to day 0, for each vaccine        strain.***Protection rate defined as the percentage of vaccinees        with a serum HI titre=40 after vaccination (for each vaccine        strain) that usually is accepted as indicating protection.

For the Cell Mediated Immune (CMI) Response

Observed Variable

At days 0 and 21: frequency of cytokine-positive CD4/CD8 cells per 10⁶in different tests. Each test quantifies the response of CD4/CD8 T cellto:

-   -   Peptide Influenza (pf) antigen (the precise nature and origin of        these antigens needs to be given/explained    -   Split Influenza (sf) antigen    -   Whole Influenza (wf) antigen.

Derived Variables:

-   -   cells producing at least two different cytokines (CD40L, IL-2,        IFNγ, TNFα)    -   cells producing at least CD40L and another cytokine (IL-2, TNFα,        IFNγ)    -   cells producing at least IL-2 and another cytokine (CD40L, TNFα,        IFNγ)    -   cells producing at least IFNγ and another cytokine (IL-2, TNFα,        CD40L)    -   cells producing at least TNFα and another cytokine (IL-2, CD40L,        IFNγ)

I.3.5.3. Analysis of Immunogenicity

The immunogenicity analysis was based on the total vaccinated cohort.For each treatment group, the following parameters (with 95% confidenceintervals) were calculated:

-   -   Geometric mean titres (GMTs) of HI and NI antibody titres at        days 0 and 21    -   Geometric mean titres (GMTs) of neutralising antibody titres at        days 0 and 21.    -   Conversion factors at day 21.    -   Seroconversion rates (SC) at day 21 defined as the percentage of        vaccinees that have at least a 4-fold increase in serum HI        titres on day 21 compared to day 0.    -   Protection rates at day 21 defined as the percentage of        vaccinees with a serum HI titre=1:40.    -   The frequency of CD4/CD8 T-lymphocytes secreting in response was        summarised (descriptive statistics) for each vaccination group,        at each timepoint (Day 0, Day 21) and for each antigen (Peptide        influenza (pf), split influenza (sf) and whole influenza (wf)).    -   Descriptive statistics in individual difference between        timepoint (Post-Pre) responses fore each vaccination group and        each antigen (pf, sf, and wf) at each 5 different tests.    -   A non-parametric test (Kruskall-Wallis test) was used to compare        the location differences between the 3 groups and the        statistical p-value was calculated for each antigen at each 5        different tests. All significance tests were two-tailed.        P-values less than or equal to 0.05 were considered as        statistically significant.

Example II Preparation of the MPL/QS21 Liposomal Adjuvant

II.3 Preparation of MPL Liquid Suspension

The MPL (as used throughout the document it is an abbreviation for3D-MPL, i.e. 3-O-deacylated monophosphoryl lipid A) liquid bulk isprepared from 3D-MPL lyophilized powder. MPL liquid bulk is a stableconcentrated (around 1 mg/ml) aqueous dispersion of the raw material,which is ready-to-use for vaccine or adjuvant formulation. A schematicrepresentation of the preparation process is given in FIG. 1.

For a maximum batch size of 12 g, MPL liquid bulk preparation is carriedover in sterile glass containers. The dispersion of MPL consists of thefollowing steps:

-   -   suspend the MPL powder in water for injection    -   desaggregate any big aggregates by heating (thermal treatment)    -   reduce the particle size between 100 nm and 200 nm by        microfluidization    -   prefilter the preparation on a Sartoclean Pre-filter unit,        0.8/0.65 μm    -   sterile filter the preparation at room temperature (Sartobran P        unit, 0.22 μm)

MPL powder is lyophilized by microfluidisation resulting in a stablecolloidal aqueous dispersion (MPL particles of a size susceptible tosterile filtration). The MPL lyophilized powder is dispersed in waterfor injection in order to obtain a coarse 10 mg/ml suspension. Thesuspension then undergoes a thermal treatment under stirring. Aftercooling to room temperature, the microfluidization process is started inorder to decrease the particle size. Microfluidization is conductedusing Microfluidics apparatus M110EH, by continuously circulating thedispersion through a microfluidization interaction chamber, at a definedpressure for a minimum amount of passages (number of cycles: n_(min)).The microfluidization duration, representing the number of cycles, iscalculated on basis of the measured flow rate and the dispersion volume.On a given equipment at a given pressure, the resulting flow rate mayvary from one interaction chamber to another, and throughout thelifecycle of a particular interaction chamber. In the present examplethe interaction chamber used is of the type F20Y Microfluidics. As themicrofluidization efficiency is linked to the couple pressure-flow rate,the processing time may vary from one batch to another. The timerequired for 1 cycle is calculated on basis of the flow rate. The flowrate to be considered is the flow rate measured with water for injectionjust before introduction of MPL into the apparatus. One cycle is definedas the time (in minutes) needed for the total volume of MPL to pass oncethrough the apparatus. The time needed to obtain n cycles is calculatedas follows:n×quantity of MPL to treat(ml)/flow rate(ml/min)

The number of cycles is thus adapted accordingly. Minimum amount ofcycles to perform (n_(min)) are described for the preferred equipmentand interaction chambers used. The total amount of cycles to run isdetermined by the result of a particle size measurement performed aftern_(min) cycles. A particle size limit (d_(lim)) is defined, based onhistorical data. The measurement is realized by photon correlationspectroscopy (PCS) technique, and is expressed as an unimodal result(Z_(average)). Under this limit, the microfluidization can be stoppedafter n_(min) cycles. Above this limit, microfluidization is continueduntil satisfactory size reduction is obtained, for maximum another 50cycles.

If the filtration does not take place immediately aftermicrofluidization, the dispersed MPL is stored at +2 to +8° C. awaitingtransfer to the filtration area.

After microfluidization, the dispersion is diluted with water forinjection, and sterile filtered through a 0.22 μm filter under laminalflow. The final MPL concentration is 1 mg/ml (0.80-1.20 mg/ml).

II.2 Preparation of MPL/QS21 Liposomal Adjuvant

This adjuvant, named AS01, comprises 3D-MPL and QS21 in a quenched formwith cholesterol, and was made as described in WO 96/33739, incorporatedherein by reference. In particular the AS01 adjuvant was preparedessentially as Example 1.1 of WO 96/33739. The AS01B adjuvant comprises:liposomes, which in turn comprise dioleoyl phosphatidylcholine (DOPC),cholesterol and 3D MPL [in an amount of 1000 μg DOPC, 250 μg cholesteroland 50 μg 3D-MPL, each value given approximately per vaccine dose], QS21[50 μg/dose], phosphate NaCl buffer and water to a volume of 0.5 ml.

The AS01E adjuvant comprises the same ingredients than AS01B but at alower concentration in an amount of 500 μg DOPC, 125 μg cholesterol, 25μg 3D-MPL and 25 μg QS21, phosphate NaCl buffer and water to a volume of0.5 ml.

In the process of production of liposomes containing MPL the DOPC(Dioleyl phosphatidylcholine), cholesterol and MPL are dissolved inethanol. A lipid film is formed by solvent evaporation under vacuum.Phosphate Buffer Saline (9 mM Na₂HPO₄, 41 mM KH₂PO₄, 100 mM NaCl) at pH6.1 is added and the mixture is submitted to prehomogenization followedby high pressure homogenisation at 15,000 psi (around 15 to 20 cycles).This leads to the production of liposomes which are sterile filteredthrough a 0.22 μm membrane in an aseptic (class 100) area. The sterileproduct is then distributed in sterile glass containers and stored in acold room (+2 to +8° C.).

In this way the liposomes produced contain MPL in the membrane (the “MPLin” embodiment of WO 96/33739).

QS21 is added in aqueous solution to the desired concentration.

Example III Pre-Clinical Evaluation of Adjuvanted and UnadjuvantedInfluenza Vaccines in Ferrets

III.1. Rationale and objectives

Influenza infection in the ferret model closely mimics human influenza,with regards both to the sensitivity to infection and the clinicalresponse.

The ferret is extremely sensitive to infection with both influenza A andB viruses without prior adaptation of viral strains. Therefore, itprovides an excellent model system for studies of protection conferredby administered influenza vaccines.

This study investigated the efficacy of various Trivalent Splitvaccines, adjuvanted or not, to reduce disease symptoms (bodytemperature) and viral shedding in nasal secretions of ferretschallenged with homologous strains.

The objective of this experiment was to demonstrate the efficacy of anadjuvanted influenza vaccine compared to the plain (un-adjuvanted)vaccine.

The end-points were:

1) Primary end-point: reduction of viral shedding in nasal washes afterhomologous challenge:

2) Secondary end-points: Analysis of the humoral response by HI titers.

III.2. Experimental Design

III.2.1. Treatment/Group (Table 1)

Female ferrets (Mustela putorius furo) aged 14-20 weeks were obtainedfrom MISAY Consultancy (Hampshire, UK). Ferrets were primed on day 0with heterosubtypic strain H1N1 A/Stockholm/24/90 (4 Log TCID₅₀/ml). Onday 21, ferrets were injected intramuscularly with a full human dose(500 μg vaccine dose, 15 μg HA/strain) of a combination of H1N1 A/NewCaledonia/20/99, H3N2 A/Panama/2007/99 and B/Shangdong/7/97. Ferretswere then challenged on day 42 by intranasal route with anheterosubtypic strain H3N2 A/Wyoming/3/2003 (4.5 Log TCID₅₀/ml).

TABLE 1 Comments Antigen(s) + Formulation + (schedule/route/ Groupdosage dosage challenge) In/Po Other treatments 1 Trivalent Full HD: 15μg IM; Day 21 In Priming H1N1 Plain HA/strain (A/Stockolm/24/90) Day 0 2Trivalent/ FullHD: 15 μg IM; Day 21 In Priming H1N1 MPL-QS21 inHA/strain (A/Stockolm/24/90) liposomes Day 0 6 ferrets/group. In/Po =Individual/pool

III.2.2. Preparation of the Vaccine Formulations (Table 2)

Formulation 1: Trivalent Split Plain (Un-Adjuvanted) Formulation:

PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well asa mixture containing Tween 80, Triton X-100 and VES (quantities takinginto account the detergents present in the strains) are added to waterfor injection. After 5 min stirring, 15 μg of each strain H1N1, H3N2 and17.5 μg of B strain are added with 10 min stirring between eachaddition. The formulation is stirred for minimum 15 minutes and storedat 4° C. if not administered directly.

Formulation 2: Trivalent Split Influenza Adjuvanted with MPL/QS21 inLiposomes:

PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well asa mixture containing Tween 80, Triton X-100 and VES (quantities takinginto account the detergents present in the strains) are added to waterfor injection. After 5 min stirring, 15 μg of each strain H1N1, H3N2 and17.5 μg of B strain are added with 10 min stirring between eachaddition. The formulation is stirred for 15 minutes. A premix of socalled “DQS21-MPLin is added to the formulation which is then stirredfor minimum 15 minutes. The DQS21-MPLin premix is a mixture of liposomes(made of DOPC 40 mg/ml, cholesterol 10 mg/ml, MPL 2 mg/ml) and theimmunostimulant QS21. This premix is incubated for a minimum of 15minutes prior to addition to the trivalent split mixture. Theconcentration of MPL and QS21 in the final formulation is 50 μg per 500μl. The formulation is stored at 4° C. if not administered directly.

Remark: In each formulation, PBS 10 fold concentrated is added to reachisotonicity and is 1 fold concentrated in the final volume. H2O volumeis calculated to reach the targeted volume.

TABLE 2 Final composition of formulations 1 and 2 (Formulations preparedwith split strains (for 500 μl)) Tween Triton X- Formulation Antigen 80100 VES DOPC Cholesterol MPL QS21 1 H1N1: 15 μg 375 μg 55 μg 50 μg — — —— H3N2: 15 μg B: 17.5 μg 2 H1N1: 15 μg 375 μg 55 μg 50 μg 1 mg 250 μg 50μg 50 μg H3N2: 15 μg B: 17.5 μg

III.2.3. Read-Outs (Table 3)

TABLE 3 Readout Timepoint Sample-type Analysis method Viral D + 1 to D +7 Post Nasal Titration shedding challenge washes Anti-HI Pre, Postpriming, Post Sera Hemagglutination antibodies immunization, Postinhibition test (HI titers) challenge

III.3. Results

A schematic representation of the results is given in FIGS. 1 and 2.

III.3.1. Humoral Immunity (FIG. 1).

Haemagglutination inhibition activity against the H3N2 vaccine strains(vaccine strain A/Panama/2007/99 and challenge strain A/Wyoming/3/2003)was detected in sera from 6 animals per group at Day 17 after intranasalheterologous priming and at Day 21 Post-immunization and Day 13Post-challenge.

Anti-Hemagglutinin antibody titers to the three influenza virus strainswere determined using the hemagglutination inhibition test (HI) asdetailed under Example 1.2.1. The conclusions are as follows:

-   -   For the two A/H3N2 strains and for all groups, a boost of HI        titers was observed in all vaccinated groups after immunization.    -   Post-immunization with A/Panama/2007/99, statistically        significant higher anti-A/Panama/2007/99 HI titers were observed        when the Trivalent Split vaccine was adjuvanted with MPL/QS21 in        liposomes compared to the Trivalent Split Plain vaccine.    -   After immunization with A/Panama/2007/99, only the Trivalent        Split adjuvanted with MPL/QS21 in liposomes was able to        significantly increase HI titers to the heterologous strain        A/Wyoming/3/2003 (cross-reactivity before challenge with this        drift strain).    -   After challenge with A/Wyoming3/2003, an significant increase of        anti-A/Wyoming/3/2003 HI titers was observed for both Trivalent        Split Plain and Trivalent Split adjuvanted with MPL/QS21 in        liposomes.    -   For A/New Caledonia/20/99 and B/Shangdong/7/97 strains,        statistically significant higher HI titers were observed when        the Trivalent Split was adjuvanted with MPL/QS21 in liposomes        compared to the Trivalent Split Plain vaccine.

III.3.2. Viral Shedding (FIG. 3).

Viral titration of nasal washes was performed on 6 animals per group asdetailed under Example 1.2.3. The nasal washes were performed byadministration of 5 ml of PBS in both nostrils in awake animals. Theinoculation was collected in a Petri dish and placed into samplecontainers at −80° C.

-   -   Two days after challenge, statistically significant lower viral        shedding was observed with Trivalent Split adjuvanted with        MPL/QS21 in liposomes compared to Trivalent Split Plain.    -   On Day 49 (7 days Post-challenge), no virus was detected in        nasal washes.

III.3.3. Conclusion of the Experiment

Higher humoral responses (HI titers) were observed with Trivalent Splitadjuvanted with MPL/QS21 in liposomes compared to the Trivalent SplitPlain for all 4 strains.

After immunization with A/Panama/2007/99, only the Trivalent Splitadjuvanted with MPL/QS21 in liposomes was able to significantly increaseHI titers to the heterologous strain A/Wyoming/3/2003 (cross-reactivitybefore challenge with this strain).

MPL/QS21 in liposomes formulations showed added benefit in terms ofprotective efficacy in ferrets (lower viral shedding after heterologouschallenge). The cross-reaction observed after immunization withTrivalent Split MPL/QS21 in liposomes against the drift strain used forthe challenge seemed to correlate with the protection effect observed inthese ferrets.

Example IV Pre-Clinical Evaluation of Adjuvanted and UnadjuvantedInfluenza Vaccines in C57BI/6 Primed Mice

IV.1. Experimental Design and Objective

C57BI/6 mice primed with heterologous strains were used for thisexperiment.

The purpose was to compare the humoral (HI titers) and CMI (ICS,intracellular cytokine staining) immune responses induced by aGlaxoSmithKline commercially available Trivalent split vaccine(Fluarix™) versus a Trivalent subunit vaccine (Chiron's vaccineAgrippal™) as well as the CMI response obtained with these vaccinesadjuvanted with Liposomes containing 3D-MPL alone, DQS21 (QS21 inliposomes, i.e. detoxified QS21) alone or MPL/QS21 in liposomes. In theexample hereinbelow, formulations were prepared starting from the splitmonobulks to reach the same composition than in the Fluarix vaccine andnot from commercially available Fluarix doses. The formulations obtainedwere called “Fluarix like”.

IV.1.1. Treatment/Group

Female C57BI/6 mice aged 6-8 weeks were obtained from Harlan Horst,Netherland. Mice were primed on day 0 with heterosubtypic strains (5 μgHA whole inactivated H1N1 A/Beijing/262/95, H3N2 A/Panama/2007/99,B/Shangdong/7/97). On day 28, mice were injected intramuscularly with1.5 μg HA Trivalent split (A/New Caledonia/20/99, A/Wyoming/3/2003,B/Jiangsu/10/2003) plain or adjuvanted (see groups in Tables 4 to 6below).

TABLE 4 Gr Antigen/Formulation Other treatment 1 Trivalent split*/Plain(un-adjuvanted) = Fluarix Heterologous like priming D 0 2 Trivalentsplit*/Liposomes containing 3D-MPL Heterologous priming D 0 3 Trivalentsplit*/DQS21 Heterologous priming D 0 4 Trivalent split*/MPL/QS21 inliposomes Heterologous priming D 0 5 Aggripal ™ (sub-unit) Heterologouspriming D 0 6 Aggripal ™ (sub-unit)/Liposomes containing 3D-Heterologous MPL priming D 0 7 Aggripal ™ (sub-unit)/DQS21 Heterologouspriming D 0 8 Aggripal ™ (sub-unit)/MPL/QS21 in liposomes Heterologouspriming D 0 9 PBS Heterologous priming D 0 *Fluarix like. 16 mice/group

IV.1.2. Preparation of the Vaccine Formulations

Formulation for Group 1:

PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well asa mixture containing Tween 80, Triton X-100 and VES (quantities takinginto account the detergents present in the strains) are added to waterfor injection. After 5 min stirring, 15 μg of each strain H1N1, H3N2 and15 μg of B strain are added with 10 min stirring between each addition.The formulation is stirred for minimum 15 minutes and stored at 4° C. ifnot administered directly.

Formulation for Group 2:

PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well asa mixture containing Tween 80, Triton X-100 and VES (quantities takinginto account the detergents present in the strains) are added to waterfor injection. After 5 min stirring, 15 μg of each strain H1N1, H3N2 and15 μg of B strain are added with 10 min stirring between each addition.The formulation is stirred for 15 minutes. Concentrated liposomescontaining 3D-MPL (made of DOPC 40 mg/ml, Cholesterol 10 mg/ml, 3D-MPL 2mg/ml) are added to reach a final MPL concentration of 50 μg per dose.The formulation is then stirred minimum 15 minutes and stored at 4° C.if not administered directly.

Formulation for Group 3:

PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well asa mixture containing Tween 80, Triton X-100 and VES (quantities takinginto account the detergents present in the strains) are added to waterfor injection. After 5 min stirring, 15 μg of each strain H1N1, H3N2 and15 μg of B strain are added with 10 min stirring between each addition.The formulation is stirred for 15 minutes. A premix made of liposomes(made of DOPC 40 mg/ml, Cholesterol 10 mg/ml) and QS21 called “DQS21” isthen added to reach a QS21 concentration of 50 μg per dose. This premixis incubated at least for 15 minutes prior to addition to the trivalentsplit mixture. The formulation is stirred for minimum 15 minutes andstored at 4° C. if not administered directly.

Formulation for Group 4:

PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well asa mixture containing Tween 80, Triton X-100 and VES (quantities takinginto account the detergents present in the strains) are added to waterfor injection. After 5 min stirring, 15 μg of each strain H1N1, H3N2 and15 μg of B strain are added with 10 min stirring between each addition.The formulation is stirred for 15 minutes. A mixture made of liposomescontaining 3D-MPL (made of DOPC 40 mg/ml, Cholesterol 10 mg/ml, 3D-MPL 2mg/ml) and QS21 is then added to reach QS21 and MPL concentrations of 50μg per dose. This mixture is incubated at least for 15 minutes prior toaddition to the trivalent split mixture. The so called “trivalent splitMPL/QS21 in liposomes” formulation is stirred for minimum 15 minutes andstored at 4° C. if not administered directly.

Remark: In groups 1 to 4, PBS 10 fold concentrated is added to reachisotonicity and is 1 fold concentrated in the final volume. H2O volumeis calculated to reach the targeted volume.

Formulation for Group 5:

One Aggripal™ dose is mixed with equal volume of PBS mod pH 7.4. Theformulation is stirred for minimum 15 minutes and stored at 4° C. if notadministered directly.

Formulation for Group 6:

PBS pH 7.4 and one Aggripal™ dose are mixed. Liposomes containing 3D-MPL(made of DOPC 40 mg/ml, Cholesterol 10 mg/ml, 3D-MPL 2 mg/ml) are thenadded under stirring to reach the equivalent of 50 μg of MPL per dose.The formulation is stirred for minimum 15 minutes and stored at 4° C. ifnot administered directly.

Remark: PBS is added to reach isotonicity in the final volume. Aggripalis half the formulation volume.

Formulation for Group 7:

PBS pH 7.4 and one Aggripal™ dose are mixed. A premix of liposomes (madeof DOPC 40 mg/ml, Cholesterol 10 mg/ml) and QS21 so called “DQS21” isthen added under stirring to reach the equivalent of 50 μg of QS21. Thispremix is incubated for at least 15 minutes prior to addition. Theformulation is stirred minimum 15 minutes and stored at 4° C. if notadministered directly.

Remark: PBS is added to reach isotonicity in the final volume. Aggripal™is half the formulation volume.

Formulation for Group 8:

PBS pH 7.4 and one Aggripal™ dose are mixed. A premix of so called“DQS21-MPLin” is added under stirring to the formulation. TheDQS21-MPLin premix is a mixture of liposomes (made of DOPC 40 mg/ml,cholesterol 10 mg/ml, MPL 2 mg/ml) and the immunostimulant QS21. Thispremix is incubated for at least 15 minutes prior to addition to theAggripal/PBS mixture. The quantity of MPL and QS21 in the formulation is50 μg each. The formulation is stirred minimum 15 minutes and stored at4° C. if not administered directly.

Remark: PBS is added to reach isotonicity in the final volume. Aggripalis half the formulation volume.

TABLE 5 Final composition of the formulations 1 to 4 prepared with splitstrains (for 1 ml) Triton Group Antigen Tween 80 X-100 VES DOPCCholesterol MPL QS21 1 H1N1: 15 μg 750 μg 110 μg 100 μg — — — — H3N2: 15μg B: 17.5 μg 2 Identical to 1 Identical to 1 110 μg 100 μg 1 mg 250 μg50 μg — 3 Identical to 1 Identical to 1 110 μg 100 μg 1 mg 250 μg — 50μg 4 Identical to 1 Identical to 1 110 μg 100 μg 1 mg 250 μg 50 μg 50 μg

TABLE 6 Final composition of the formulations 5 to 8 prepared withAggripal ™ vaccine (1 ml) Group Antigen DOPC Cholesterol MPL QS21 51dose of Aggripal — — — — vaccine 6 Identical to 5 1 mg 250 μg 50 μg — 7Identical to 5 1 mg 250 μg — 50 μg 8 Identical to 5 1 mg 250 μg 50 μg 50μg

IV.1.3. Read-Outs (Table 7)

TABLE 7 Sample Read-out Timepoint type In/Po Analysis method Anti-HI Day21 Post- Sera In Hemagglutination antibodies Immunization Inhibitiontest (HI titers) (Day 49) CD4, CD8, Day 7 Post- PBLs Po Intracellularcytokine IL-2, IFN-γ Immunization staining (ICS) (FACS) (Day 35) In =Individual/Po = Pool

IV. 2. Results

IV.2.1. Humoral Response (HI Titers 21 Days Post Immunization).

Humoral Responses by HI Titers—FIG. 4.

Haemagglutination inhibition activity against the three vaccine strains(A/New Caledonia/20/99, A/Wyoming/3/2003, B/Jiangsu/10/2003) wasdetected in sera from 8 animals per group at Day 21 Post-immunization.

-   -   Compared to mice immunized with PBS, an increase in HI titers        was observed after immunization with all Flu vaccine candidates        tested for all three strains (Trivalent split or Trivalent        subunit vaccine).    -   For all three strains, statistically significant higher HI        titers were observed in mice immunized with Trivalent split        adjuvanted with DQS21 alone or MPL/QS21 in liposomes compared to        mice immunized with Trivalent split Flu plain or adjuvanted with        Liposomes containing 3D-MPL alone. The ranking for the humoral        response was as follow: (MPL/QS21 in liposomes=DQS21        alone)>(Liposomes containing 3D-MPL alone=Plain)>PBS    -   For all three strains, statistically significant higher HI        titers were observed in mice immunized with Trivalent subunit        adjuvanted with DQS21 alone, Liposomes containing 3D-MPL alone        or MPL/QS21 in liposomes compared to mice immunized with        Trivalent split plain. The ranking for the humoral response was        as follow: (MPL/QS21 in liposomes=DQS21 alone=Liposomes        containing 3D-MPL alone)>Plain>PBS.    -   Trivalent split and Trivalent subunit induced similar HI titers        when formulations were not adjuvanted or adjuvanted with DQS21        alone or MPL/QS21 in liposomes.

IV.2.2. Cell-Mediated Immune Response (ICS at Day 7 Post Immunization).

CD4 T Cell Responses—FIG. 5

PBMCs from 8 mice per group were harvested at Day 7 Post-immunizationand tested in 4 pools of 2 mice/group.

In terms of Flu whole virus-specific total CD4+ T cells (expressingIL-2, IFN-γ and both cytokines):

-   -   Whatever the formulation, identical CD4+ T cell responses were        observed between the Trivalent split and Trivalent subunit        vaccines.    -   Higher CD4+ T cell responses were observed for Trivalent        formulations (split or subunit) adjuvanted with MPL/QS21 in        liposomes when compared to Trivalent formulations (split or        subunit) plain or adjuvanted with Liposomes containing 3D-MPL        alone or DQS21 alone.    -   For the cellular response induced by a Trivalent formulation        (split or subunit), there is a synergic effect of Liposomes        containing 3D-MPL+DQS21 compared to DQS21 alone or Liposomes        containing 3D-MPL alone.    -   The ranking for the cellular response was as follow: MPL/QS21 in        liposomes>(Liposomes containing 3D-MPL alone=DQS21        alone=Plain=PBS).

IV.3. Summary of Results and Conclusions

-   -   For all three strains, statistically significant higher HI        titers were observed in mice immunized with Trivalent        formulations (split or subunit) adjuvanted with DQS21 alone or        MPL/QS21 in liposomes compared to mice immunized with Trivalent        formulations (split or subunit) plain. Liposomes containing        3D-MPL alone seemed to induced higher humoral response when        formulated with Trivalent subunit than Trivalent split.    -   Whatever the formulation, similar CD4+ T cell responses were        obtained for Trivalent split (Fluarix) and Trivalent subunit        (Agrippal).    -   Trivalent formulations (split or subunit) adjuvanted with        MPL/QS21 in liposomes induced higher CD4+ T cell responses        compared to Trivalent formulations (split or subunit) plain or        adjuvanted with Liposomes containing 3D-MPL alone or QS21 in        liposomes (DQS21) alone.

Example V Preclinical Comparison of a Vaccine Containing a SplitInfluenza Antigen Preparation Adjuvanted with 3D-MPL/QS21 in a LiposomalFormulation (3D-MPL at Two Different Concentrations)

V.1—Mice.

V.1.1—Experimental Design and Objective.

C57B1/6 mice primed with heterologous strains were used for thisexperiment. The purpose was to analyse the humoral (HI titers) and CMI(ICS, intracellular cytokine staining) immune responses induced by aGlaxoSmithKline commercially available Trivalent split vaccine(Fluarix™) in un-adjuvanted form, and when adjuvanted with liposomescontaining two different concentrations of 3D-MPL and QS21.

V.1.2 Treatment/Group

Female C57B1/6 mice aged 8 weeks were obtained from Harlan Horst,Netherlands. Mice were primed intranasally on day 0 with heterosubtypicstrains (whole inactivated A/Beijing/262/95, H3N2 A/Panama/2007/99,B/Shandong/7/97). On day 28, mice were injected intramuscularly withTrivalent Split (A/New Caledonia,A/Wyoming, B/Jiangsu) plain oradjuvanted with two different concentrations of immunostimulants inliposomal formulations (see groups in table 8 below).

TABLE 8 Antigen(s) + Formulation + Group dosage dosage Other treatments1 Trivalent Split Flu - Plain Heterologous priming 1.5 μg/strain/50 μl D0 whole inactivated 5 μg/20 μl intranasally 2 Trivalent Split Flu -Liposomes Heterologous priming 1.5 μg/strain/50 μl containing D 0 wholeinactivated 3D-MPL 5 μg/20 μl intranasally 50 μg per 0.5 ml dose 3Trivalent Split Flu - DQS21 Heterologous priming 1.5 μg/strain/50 μl 50μg per 0.5 D 0 whole inactivated ml dose 5 μg/20 μl intranasally 4Trivalent Split Flu - MPL and QS21 Heterologous priming 1.5 μg/strain/50μl 25 μg per 0.5 D 0 whole inactivated ml dose 5 μg/20 μl intranasally 5Trivalent Split Flu - MPL and QS21 Heterologous priming 1.5 μg/strain/50μl 50 μg per 0.5 D 0 whole inactivated ml dose 5 μg/20 μl intranasally 6PBS None Heterologous priming D 0 whole inactivated 5 μg/20 μlintranasally

Formulations were prepared as in example IV.

V.1.3—Results.

Humoral Responses by HI Titers—FIG. 24.

Hemagglutination inhibition activity against the 3 vaccine strains wasdetected in sera from 9 animals/group on day 21 post immunisation.

-   -   Compared to mice immunized with PBS, an increase in HI titres        was observed after immunization with all Flu vaccine candidates        tested for all three strains.    -   For all three strains, statistically significant higher HI        titers were observed in mice immunized with Trivalent Split        adjuvant with MPL and QS21 at either concentration compared to        mice immunized with the Trivalent Flu Split Plain (p value        max=0.03)    -   No statistically significant difference was observed between the        two liposomal adjuvant groupsadjuvant groups

Cell-Mediated Immune Response (ICS at Day 7 Post-Immunisation)—FIG. 25.

PBMC's from 9 mice/group were harvested 7 days post-immunisation andtested in three pools of 3 mice/group. In terms of whole Fluvirus-specific CD4+ T cells expressing IL-2, IFN-γ or both cytokines:

As can be seen from FIG. 25 the highest IFN-γ CD4+ T cell-specificresponses were obtained after immunization with trivalent splitadjuvanted with the highest concentration of immunostimulants. However,IL2 and IL2+ IFN-γ T cell responses were similar between the twoconcentrations of immunostimulants.

Example VI Clinical Trial in an Elderly Population Aged Over 65 Yearswith a Vaccine Containing a Split Influenza Antigen PreparationAdjuvanted with MPL/QS21 in a Liposomal Formulation (3D-MPL at TwoDifferent Concentrations)

VI.1. Study Design and Objectives

An open, randomized phase I/II study to demonstrate the non inferiorityin term of cellular mediated immune response of GlaxoSmithKlineBiologicals influenza candidate vaccines containing various adjuvantsadministered in elderly population (aged 65 years and older) as comparedto Fluarix™ (known as α-Rix™ in Belgium) administered in adults (18-40years)

Four parallel groups were assigned:

-   -   75 adults (aged 18-40 years) in one control group receiving one        dose of Fluarix™

(Fluarix group)

-   -   200 elderly subjects (aged 65 years and older) randomized 3:3:2        into three groups:        -   one group with 75 subjects receiving influenza vaccine            adjuvanted with AS01B    -   One group with 75 subjects receiving influenza vaccine        adjuvanted with AS01E        -   Reference Flu group with 50 subjects receiving one dose of            Fluarix™

Primary Objective

The primary objective is to demonstrate the non inferiority 21 dayspost-vaccination of the influenza adjuvanted vaccines administered inelderly subjects (aged 65 years and older) as compared to Fluarix™administered in adults (aged 18-40 years) in terms of frequency ofinfluenza-specific CD4 T-lymphocytes producing at least two differentcytokines (CD40L, IL-2, TNF-α, IFN-γ).

Secondary Objectives

The secondary objectives are:

1) To evaluate the safety and reactogenicity of vaccination withcandidate influenza vaccines adjuvanted during 21 days following theintramuscular administration of the vaccine in elderly subjects (aged 65years and older). Fluarix™ is used as reference.

2) To evaluate the humoral immune response (anti-haemagglutinin titre)21, 90 and 180 days after vaccination with influenza candidate vaccinesadjuvanted. Fluarix™ is used as reference.

Tertiary Objective

The tertiary objective is to evaluate the cell mediated immune response(production of IFN-γ, IL-2, CD40L, and TNF-α and memory B-cell response)21, 90 and 180 days after vaccination with adjuvantedinfluenza-vaccines. Fluarix™ is used as reference.

VI.2. Vaccine Composition and Administration

Two different adjuvants have been used:

-   -   1. AS01B a liposome-based adjuvant containing 50 μg MPL and QS21    -   2. AS01E a two-fold diluted formulation of AS01B

Control: full dose of Fluarix™ by IM administration.

All vaccines are intended for intramuscular administration. The strainsused in the five vaccines are the ones that have been recommended by theWHO for the 2005-2006 Northern Hemisphere season, i.e. A/NewCaledonia/20/99 (H1N1), A/New York/7/2004 (H3N2) and B/Jiangsu/10/2003.

The three inactivated split virion antigens (monovalent bulks) used informulation of the adjuvanted influenza candidate vaccine, are exactlythe same as the active ingredients used in formulation of the commercialFluarix™/α-Rix™—GSK Bio's split virion inactivated influenza vaccine.They are derived from egg-grown viruses. The influenza strains are therecommended ones for the 2005/2006 season, as used in the formulation ofthe commercial Fluarix™/α-Rix™ 2005/2006.

The strains used in the three vaccines are the ones that have beenrecommended by the WHO for the 2005-2006 Northern Hemisphere season i.e.

-   -   A/New Caledonia/20/99 (H₁N₁) IVR-116    -   A/New York/55/2004 (H3N2) NYMC X-157    -   B/Jiangsu/10/2003

Like Fluarix™/α-Rix™ the adjuvanted vaccine contains 15 μghaemagglutinin (HA) of each influenza virus strain per dose.

VI.2.1. Description of the AS01B Adjuvanted Vaccine Lots

The AS01B-adjuvanted influenza candidate vaccine is a 2 componentsvaccine consisting of a concentrated trivalent inactivated split virionantigens presented in a glass vial and of a glass vial containing theAS01B adjuvant. At the time of injection, the content of the adjuvantvial is withdrawn and injected into the vial that contains theconcentrated trivalent inactivated split vrion antigens. After mixingthe content is withdrawn into the syringe and the needle is replaced byan intramuscular needle. The used needle is replaced by an intramuscularneedle and the volume is corrected to 1 ml. One dose of thereconstituted AS01B-adjuvanted influenza candidate vaccine correspondsto 1 mL.

The AS01B-adjuvanted influenza candidate vaccine is a preservative-freevaccine.

VI.2.2. Composition of the AS01B adjuvanted clinical lot

One dose of the reconstituted AS01B-adjuvanted influenza vaccinecorresponds to 1 mL. Its composition is given in Table 8. It contains 15μg HA of each influenza virus strain as in the registeredFluarix™/α-Rix® vaccine.

TABLE 8 Composition (influenza and adjuvant components) of thereconstituted AS01B adjuvanted influenza candidate vaccine AnalyticalComponent Quantity per dose Reference ACTIVE INGREDIENTS Inactivatedsplit virions A/New Caledonia/20/99 (H1N1) 15 μg HA Ph. Eur. 158 IVR-116A/New York/55/2004 (H3N2) 15 μg HA Ph. Eur. 158 NYMC X-157B/Jiangsu/10/2003 15 μg HA Ph. Eur. 158 AS01B ADJUVANT Liposomes dioleylphosphatidylcholine 1000 μg GSK Bio 3217 (DOPC) Cholesterol 250 μg Ph.Eur. 0993 MPL 50 μg GSK Bio 2972 QS21 50 μg GSK Bio 3034

VI.2.3. Production Method of the AS01B Adjuvanted Vaccine Lot

The manufacturing of the AS01B-adjuvanted influenza vaccine consists ofthree main steps:

-   -   Formulation of the trivalent final bulk (2× concentrated)        without adjuvant and filling in the antigen container    -   Preparation of the AS01B adjuvant    -   Extemporaneous reconstitution of the AS01B adjuvanted split        virus vaccine.

Formulation of the Trivalent Final Bulk without Adjuvant and Filling inthe Antigen Container

The volumes of the three monovalent bulks are based on the HA contentmeasured in each monovalent bulk prior to the formulation and on atarget volume of 1320 ml. Concentrated phosphate buffered saline PO4Na/K₂ (80 μl/dose) and a pre-mixture of Tween 80, Triton X-100 anda-tocopheryl hydrogen succinate are diluted in water for injection. Thethree concentrated monobulks (A/New Caledonia/20/99 IVR-116, A/NewYork/55/2004 NYMC X-157, B/Jiangsu/10/2003) are then successivelydiluted in the resulting phosphate buffered saline/Tween 80-TritonX-100-α-tocopheryl hydrogen succinate solution (pH 7.8, 81 mM NaCl, 1.56mM KCl, 4.79 mM Na₂HPO₄, 0.87 mM KH₂PO₄, 7.2 mM NaH2PO4, 72.8 mM K2HPO4,750 μg/ml Tween 80, 110 μg/ml Triton X-100 and 100 μg/ml α-tocopherylhydrogen succinate) in order to have a final concentration of 30 μg HAof A (H1N1 and H3N2) strains per ml of trivalent final bulk (15 μg HA/Astrain/500 μl trivalent final bulk) and 35 μg HA of B strain (17.5 μgHA/B strain/500 μl trivalent final bulk). Between addition of eachmonovalent bulk, the mixture is stirred for 10-30 minutes at roomtemperature. After addition of the last monovalent bulk and 15-30minutes of stirring, the pH is checked and adjusted to 7.65±0.25 withHCl or NaOH.

The trivalent final bulk of antigens is aseptically filled into 3-mlsterile Type I (Ph. Eur.) glass vials. Each vial contains a volume of600 μl (500 μl+100μ, overfill).

Preparation of AS01B Adjuvant Bulk and Filling in the Adjuvant Container

The adjuvant AS01B is prepared by mixing of two components: QS21 andliposomes containing MPL. The preparation of each of these components issummarized below. QS21 is a triterpene glycoside, obtained from the treebark of Quillaja saponaria, and is produced by Aquila Worchester, Mass.,USA (now Antigenics).

QS21 is provided to GSK Biologicals as a lyophillised powder. Thepreparation of QS21 at GSK Bio consists of suspension of QS21 powder inwater for injection at a concentration of approximately 5 mg/mL, pHadjustment to pH 6.0±0.2 and sterile filtration. The liquid bulk QS21 isstored at −20° C. in polyethylene containers.

MPL is the 3-O-deacyl-4′-monophosphoryl lipid A obtained by sequentialacid and base hydrolyses of the lipopolysaccharide from the Re595 strainof Salmonella minnesota. It is produced by GSK Biologicals, Hamilton,Mont. Bulk MPL is supplied as the lyophilized salt of triethylamine(TEA).

In the process of production of MPL-containing liposomes, DOPC (Dioleylphosphatidylcholine), cholesterol and MPL are dissolved in ethanol. Alipid film is formed by solvent evaporation under vacuum. PhosphateBuffer Saline made of 9 mM Na₂HPO₄, 41 mM KH₂PO₄, 100 mM NaCl at pH 6.1is added and the mixture is submitted to prehomogenization followed byhigh pressure homogenization at 15,000 psi (+/−20 cycles). This leads tothe production of liposomes, which are sterile filtered through a 0.22μm membrane in an aseptic (class 100) area. The sterile product is thendistributed in sterile glass containers and stored in the cold room (+2to +8° C.).

Sterile bulk preparation of liposomes is mixed with sterile QS21 bulksolution. After 30 min stirring, this mixture is added to a mixture ofwater for injection and phosphate 500 mM, NaCl 1M pH 6.1 when diluted 10times. Quantity of the phosphate 500 mM, NaCl 1M pH 6.1 when diluted 10times, is calculated to reach isotonicity in the final volume. The pH ischecked. The adjuvant is then sterile filtered (0.22 μm) and asepticallydistributed into vials. The vials are stored at +2 to +8° C.

The AS01B diluent is an opalescent colorless liquid, free from foreignparticles, contained in a sterile, type 1 glass vial. The target fillfor each vial is 0.7 ml in order to meet the specification (≥0.5 ml).

Extemporaneous Reconstitution of the AS01B Adjuvanted Split VirusVaccine

At the time of injection, the content of the vial containing theadjuvant is withdrawn and is injected into the vial that contains theconcentrated trivalent inactivated split vrion antigens. After mixing,the content is withdrawn into the syringe and the needle is replaced byan intramuscular needle, and the volume is corrected to 1 ml. One doseof the reconstituted AS01B-adjuvanted influenza candidate vaccinecorresponds to 1 mL.

VI.2.4. Description of the AS01E Adjuvanted Vaccine Lots

The AS01E adjuvanted influenza candidate vaccine is a 3 componentsvaccine consisting of a concentrated trivalent inactivated split virionantigens presented in a glass vial, a glass vial containing the AS01Badjuvant and a glass vial containing the diluent (sodium chloridesolution for injection) for the two-fold dilution of AS01B.

To prepare the AS01E adjuvant the content of the diluent vial iswithdrawn with a syringe and injected into the vial containing the AS01Badjuvant, followed by mixing. At the time of injection, 600 μl AS01Eadjuvant is withdrawn with a syringe from the AS01E vial and injectedinto the vial that contains the concentrated trivalent inactivated splitvrion antigens. After mixing the content is withdrawn into the syringeand the needle is replaced by an intramuscular needle. One dose of thereconstituted AS01B-adjuvanted influenza candidate vaccine correspondsto 1 mL.

The AS01E-adjuvanted influenza candidate vaccine is a preservative-freevaccine.

VI.2.5. Composition of the AS01E Adjuvanted Clinical Lot

One dose of the reconstituted AS01E-adjuvanted influenza vaccinecorresponds to 1 mL. Its composition is given in Table 9. It contains 15μg HA of each influenza virus strain as in the registeredFluarix™/α-Rix® vaccine.

TABLE 9 Composition (influenza and adjuvant components) of thereconstituted AS01E adjuvanted influenza candidate vaccine AnalyticalComponent Quantity per dose Reference ACTIVE INGREDIENTS Inactivatedsplit virions A/New Caledonia/20/99 (H1N1) 15 μg HA Ph. Eur. 158 IVR-116A/New York/55/2004 (H3N2) 15 μg HA Ph. Eur. 158 NYMC X-157B/Jiangsu/10/2003 15 μg HA Ph. Eur. 158 AS01B ADJUVANT Liposomes dioleylphosphatidylcholine 500 μg GSK Bio 3217 (DOPC) Cholesterol 125 μg Ph.Eur. 0993 MPL 25 μg GSK Bio 2972 QS21 25 μg GSK Bio 3034

VI.2.6. Production Method of the AS01E Adjuvanted Vaccine Lot

The manufacturing of the AS01B-adjuvanted influenza vaccine consists ofthree main steps:

-   -   Formulation of the trivalent final bulk (2× concentrated)        without adjuvant and filling in the antigen container    -   Preparation of the AS01B adjuvant    -   Preparation of the AS01E adjuvant followed by extemporaneous        reconstitution of the AS01E adjuvanted split virus vaccine.

Formulation of the Trivalent Final Bulk without Adjuvant and Filling inthe Antigen Container

Reference is made to section V.2.3 for the AS01B adjuvanted influenzavaccine.

Preparation of AS01B Adjuvant Bulk and Filling in the Adjuvant Container

Reference is made to section V.2.3 for the AS01B adjuvanted influenzavaccine.

Extemporaneous Reconstitution of the AS01E Adjuvanted Split VirusVaccine

To prepare the AS01E adjuvant the content of the diluent vial iswithdrawn with a syringe and injected into the vial containing the AS01Badjuvant, followed by mixing. At the time of injection, 600 μl AS01Eadjuvant is withdrawn with a syringe from the AS01E vial and injectedinto the vial that contains the concentrated trivalent inactivated splitvirion antigens. After mixing the content is withdrawn into the syringeand the needle is replaced by an intramuscular needle. One dose of thereconstituted AS01E-adjuvanted influenza candidate vaccine correspondsto 1 mL.

Four scheduled visits per subject: at days 0, 21, 90 and 180 with bloodsample collected at each visit to evaluate immunogenicity.

Vaccination schedule: one injection of influenza vaccine at day 0

VI.2.7—Immunological Assays

Haemagglutination—inhibition assay

The immune response is determined by measuring Haemagglutinationinhibition (HI) antibodies using the method described by the WHOcollaborating Centre for influenxa, Centres for Diseases Control,Atlanta, USA (1991). Antibody titre measurements were conducted onthawed frozen serum samples with a standardised and comprehensivelyvalidated micromethod using 4 haemagglutination-inhibiting units (4 HIU)of the appropriate antigens and a 0.5% fowl erythrocyte suspension.Non-specifric serum inhibitors were removed by heat treatment andreceptor-destroying enzyme. The sera obtained were evaluated for HIantibody levels. Starting with an initial dilution of 1:10, a dilutionseries (by a factor of 2) was prepared up to an end dilution of 1:20480.The titration end-point was taken as the highest dilution step thatshows complete inhibition (100%) of haemagglutination. All assays wereperformed in duplicate.

Cytokine Flow Cytometry)CFC) used to evaluate the frequency ofcytokine(s)-positive CD4 or CD8 T lymphocytes.

Peripheral blood antigen-specific CD4 and CD8 T cells can berestimulated in vitro to produce CD40L, IL-2, TNF-α and IFN-γ ifincubated with their corresponding antigen. Consequently,antigen-specific CD4 and CD8 T cells can be enumerated by flow cytometryfollowing conventional immunofluorescence labelling of cellularphenotype as well as intracellular cytokines production. In the presentstudy, influenza vaccine antigens will be used as antigens torestimulate influenza-specific T cells. Results will be expressed as afrequency of cytokine(s)-positive CD4 or CD8 T cell within the CD4 orCD8 T cell sub-population.

ELISPOT Used to Evaluate Frequency of Memory B-Cell

The B cell Elispot technology allows the quantification of memory Bcells specific to a given antigen. Memory B cells can be induce todifferentiate into plasma cells in-vitro following cultivation with CpGfor 5 days. In-vitro generated antigen-specific plasma cells cantherefore be enumerated using the B-cell elispot assay. Briefly,in-vitro generated plasma cells are incubated in culture plates coatedwith antigen. Antige-specific plasma cells will form antibody/antigenspots, which can be detected by conventional immuno-enzymatic procedure.In the present study, influenza vaccine strains or anti-humanimmunoglobulin are used to coat culture plates in order to enumerateanti-influenza or IgG secreting plasma cells, respectively. Results areexpressed as a frequency of antigen-specific plasma within a million ofIgG-producing plasma cells.

Exploratory Characterisation of PBMCs

The expression of selected surfact/activation markers (i.e. CD4., CD8,CD45RO, CD45 RA, CD28, CD27 or some KIR) can be performed. The functionof vaccine-induced T lymphocytes can be addressed by the analysis ofhoming markers (i.e. CCR7, CXCR5), of cytokines (T helper 1 or T helper2 cytokines), or by analysing the expression of factors associated withregulatory functions such as Foxp3, CTLA-4, or TGFβ. In particular, theCD8+CD28− population or other regulatory T cell populations can beanalysed in relation the humoral, B and T cell responses to the vaccineantigen.

VI.3. Immunogenicity Results

VI.3.1. CMI Endpoints and Results

In order to characterize the CMI response after vaccination with theadjuvanted influenza vaccines, CD4 and CD8 T-lymphocytes wererestimulated in vitro using antigens from the three vaccine strains(used individually or pooled). Influenza-specific CD4/CD8 T-lymphocyteswere enumerated by flow cytometry following conventionalimmunofluorescence labelling of intracellular cytokines production(IL-2, IFN-γ, TNF-α and CD40L).

Evaluation of the Primary Endpoint.

At day 21: CMI response in all subjects in terms of frequency ofinfluenza-specific CD4 T-lymphocyte per 10⁶ in tests producing at leasttwo different cytokines (IL-2, IFN-γ, TNF-α and CD40L).

For the evaluation of CMI response, frequency of influenza-specific CD4are analysed as follows:

Using the non-inferiority approach, the non inferiority of at least oneinfluenza adjuvanted candidate vaccine (administered to elderly aged ≥65years—the group termed Flu elderly or Flu ELD) compared to Fluarix™(administered to adults aged 18-40 years—the group termed Flu Young orFlu YNG) was reached when the upper limit of two-sided 98.75% confidenceinterval on Geometric Mean (GM) ratio (between the Fluarix™ (18-40years) group and the influenza adjuvanted candidate vaccine 65 yearsgroup) in terms of frequency of influenza-specific CD4 T-cells producingat least two cytokines at day 21) was below 2.0

${{UL}_{98.75\%\mspace{14mu}{CI}}\left( \frac{{GM}_{{Fluarix}\mspace{14mu}{adults}}}{G\; M_{influenzaAdjuvanted}} \right)} < 2$

The 98.75% CI of GM ratios, 21 days after vaccination, was computedusing an analysis of covariance (ANCPVA) model on the logarithm 10transformation of the frequences. The ANCOVA model included the vaccinegroup as fixed effect (Fluarix™ (18-40 years) versus the influenzaadjuvanted candidate vaccine 65 years)) and the pre-vaccinationfrequency as a regressor. The GM ratio and their 98.75% CI are derivedas exponential-transformation of the corresponding group contrast in themodel. The 98.75% CI for the adjusted GM is obtained byexponential-transformation of the 98.75% CI for the group least squaremean of the above ANCOVA model.

Results—Inferential Analysis (Table 10)

The adjusted GM and GM ratios (with their 98.75% CI) ofinfluenza-specific CD4 T-lymphocyte producing at least two cytokines(IL-2, IFN-γ, TNF-α and CD40L) at day 21, after in vitro restimulationwith “pooled antigens II”, are presented in Table 10. For eachadjuvanted influenza vaccine, the upper limit of two-sided 98.75% CI ofGM ratio is far below the clinical limit of 2.0. This shows thenon-inferiority of both adjuvanted influenza vaccines administered toelderly subjects compared to the Fluarix™ vaccine administered in adultsaged between 18 and 40 years in term of post-vaccination frequency ofinfluenza-specific CD4.

TABLE 10 Adjusted GM ratio of influenza-specific CD4 T cellsproducing atleast two cytokines after restimulation with pooled vaccine antigens,Day 21 (ATP cohort for immunogenicity) GM ratio (Flu YNG/AS01B) Flu YNGAS01B 98.8% CI N GM N GM Value LL UL 74 2844.8 71 2725.6 1.04 0.79 1.38GM ratio (Flu YNG/AS01E) Flu YNG AS01E 98.8% CI N GM N GM Value LL UL 742879.6 74 2697.0 1.07 0.79 1.44 Adjusted GM = geometric mean antibodyadjusted for baseline titre; N = Number of subjects with both pre- andpost-vaccination results available; 98.8% CI = 98.8% confidence intervalfor the adjusted GM ratio (Ancova model: adjustment for baseline); LL =lower limit, UL = upper limit; Data source = Appendix table IIIA

Results—Descriptive Analysis (FIG. 6)

The main findings were:

1) Before vaccination the CMI response is higher in young adults than inelderly

2) After vaccination:

-   -   there was a booster effect of the influenza vaccine on the CMI        response in young adults (18-40 years)    -   the CMI response in the elderly having received the adjuvanted        influenza vaccine is comparable to the CMI response of young        adults.

The difference between pre and post-vaccination in CD4 T-lymphocytesresponses for all cytokines investigated (IL-2, CD40L, TNF-α and IFN-γ)was significantly higher with the adjuvanted vaccines compared toFluarix™ for all tests.

Analysis of the Tertiary Objective:

In order to evaluate the tertiary end point, the frequency ofinfluenza-specific CD4/CD8 T-lymphocytes and memory B-cells weremeasured at days 0, 21, 90 and 180.

-   -   The frequency of influenza-specific cytokine-positive CD4/CD8        T-lymphocytes was summarised (descriptive statistics) for each        vaccination group at days 0 and 21, for each antigen.    -   A Non-parametric test (Wilcoxon test) was used to compare the        location of difference between the two groups (influenza        adjuvanted vaccine versus Fluarix™) and the statistical p-value        is calculated for each antigen at each different test.    -   Descriptive statistics in individual difference between day        21/day 0 (Post-/Pre-vaccination) responses is calculated for        each vaccination group and each antigen at each different test.    -   A Non-parametric test (Wilcoxon test) is used to compare the        individual difference Post-/Pre-vaccination) and the statistical        p-value will be calculated for each antigen at each different        test.

The p-values from Wilcoxon test used to compare the difference in thefrequency of influenza-specific CD4 T-lymphocytes are presented in Table11.

Results—Evaluation of the Tertiary End-Point (Table 11)

The main conclusions are:

-   -   Pre-vaccination GM frequencies of influenza-specific CD4 T cells        were similar in all groups of elderly subjects but superior in        the adults aged between 18 and 40 years.    -   In elderly subjects, post-vaccination (day 21) frequency of        influenza-specific CD4 T lymphocytes was significantly higher        after vaccination with adjuvanted vaccines than with Fluarix™    -   Post-vaccination frequency of influenza-specific CD4 T        lymphocytes remained lower in elderly subjects vaccinated with        AS01B or AS01E adjuvanted vaccines than in adults aged between        18 and 40 years vaccinated with Fluarix™    -   Pre-vaccination and post vaccination GM frequency of        influenza-specific CD8 T cell was essentially similar in all        groups.

TABLE 11 Inferential statistics: p-values from Kruskal-Wallis Tests forCD4 T cells at each time point (ATP Cohort for immunogenicity) P-valueAS01B vs. Flu YNG AS01E vs. Flu YNG Test description day 0 day 21 day 0day 21 ALL DOUBLES <0.0001 0.0070 <0.0001 0.0025 CD4OL <0.0001 0.0056<0.0001 0.0015 IFNγ <0.0001 0.0009 <0.0001 0.0006 IL2 <0.0001 0.0029<0.0001 0.0021 TFNα <0.0001 0.0295 <0.0001 0.0378 AS01B vs. Flu ELDAS01E vs. Flu ELD day 0 day 21 day 0 day 21 ALL DOUBLES 0.6165 0.00040.8744 0.0018 CD4OL 0.7560 0.0005 0.9504 0.0021 IFNγ 0.9936 0.00080.9835 0.0029 IL2 0.6702 0.0011 0.7855 0.0023 TFNα 0.5450 0.0022 0.66880.0040

Results—Evaluation of the Humoral Immune Response Endpoints

Observed Variables:

At days 0, 21, 90 and 180: serum haemagglutination-inhibition (HI)antibody titres, tested separately against each of the three influenzavirus strains represented in the vaccine (anti-H1N1, anti-H3N2 &anti-B-antibodies).

The cut-off value for HI antibody against all vaccine antigens wasdefined by the laboratory before the analysis (and equals 1:10). Aseronegative subject is a subject whose antibody titre is below thecut-off value. A seropositive subject is a subject whose antibody titreis greater than or equal to the cut-off value. Antibody titre below thecut-off of the assay is given an arbitrary value of half the cut-off.

Based on the HI antibody titres, the following parameters arecalculated:

-   -   Geometric mean titres (GMTs) of HI antibody at days 0 and 21,        calculated by taking the anti-log of the mean of the log titre        transformations.    -   Seroconversion factors (SF) at day 21 defined as the fold        increase in serum HI GMTs on day 21 compared to day 0.    -   Seroconversion rates (SC) at day 21 defined as the percentage of        vaccinees with either a pre-vaccination HI titre<1:10 and a        post-vaccination titre 1:40, or a pre-vaccination titre 1:10 and        a minimum 4-fold increase at post-vaccination titre.    -   Seroprotection rates (SPR) at day 21 defined as the percentage        of vaccinees with a serum HI titre≥1:40.

The 95% CI for GM is obtained within each group separately. The 95% CIfor the mean of log-transformed titre is first obtained assuming thatlog-transformed titres are normally distributed with unknown variance.The 95% CI for the GM is then obtained by exponential-transformation ofthe 95% CI for the mean of log-transformed titre.

Missing serological result for a particular antibody measurement is notreplaced. Therefore a subject without serological result at a given timepoint do not contribute to the analysis of the assay for that timepoint.

Humoral Immune Response Results (FIG. 7 and Table 12)

Pre-vaccination GMTs of HI antibodies for all 3 vaccine strains werewithin the same range in the 4 treatment groups. After vaccination,there is clear impact of the 2 adjuvants which increase the humoralresponse in elderly, compared to standard Fluarix in the same population(FIG. 7, shown on a linear scale, but same impact obviously seen ifshown on a logarithmic scale).

GMTs are

-   -   significantly higher for H1 N1 for AS01E    -   significantly higher for H3N2 for both adjuvants.    -   No significant difference was observed in terms of        post-vaccination GMTs between the two groups of subjects having        received the adjuvanted vaccines.

Twenty one days after vaccination, the subjects of Fluarix (18-40 years)had a higher HI response for New Caledonia and B/Jangsu strains.

As shown in Table 12 the adjuvanted influenza vaccines exceeded therequirements of the European authorities for annual registration ofsplit virion influenza vaccines [“Note for Guidance on Harmonization ofRequirements for Influenza Vaccines for the immunological assessment ofthe annual strain changes” (CPMP/BWP/214/96)] in subjects aged over 60years.

After vaccination, there was a statistically difference in terms ofseroprotection rates of HI antibodies between Fluarix (A5 years) groupand

-   -   Flu/AS01B and Flu/AS01E for A/H1N1/New Caledonia strain

For each vaccine strain, the seroprotection rates for the 2 influenzaadjuvanted vaccine groups are in the same range compared to Fluarix(18-40 years) group.

For each vaccine strain, the seroconversion rates for the 2 influenzaadjuvanted vaccine groups are in the same range compared to Fluarix(18-40 years) group excepted for New Caledonia strain.

TABLE 12 Seroprotection rates seroconversion rates and conversionfactors at day 21 (ATP cohort for immunogenicity) Seroconversion rateSeroprotection rate (≥4-fold increase) Conversion factor Strains Group N(HI titre ≥40) % [95% CI] % [95% CI] % EU standard (>60years) >60% >30% >2.0 EU standard (<60 years) >70% >40% >2.5 A/New FluYng 75 100   77.3 35.1  Caledonia [95.20-100]  [66.2-86.2]  {circumflexover ( )}21.9-56.4] (H1N1) Flu Elderly 49 71.4 30.6 3.7 [56.74-83.42][18.3-5.4]  [2.4-5.7] FluAS01B 75 97.3 48.0 4.5 [90.70-99.68][36.5-59.8] [3.3-6.1] FluAS01E 75 93.3 52.0 5.0 [85.12-97.80][40.2-63.7] [3.6-6.9] A/New York Flu Yng 75 93.3 76.0 9.2 (H3N2)[85.12-97.80] [64.7-85.1]  [7.1-11.8] Flu Elderly 49 81.6 69.4 8.2[67.98-91.24] [54.6-81.7]  [5.7-11.8] FluAS01B 75 96.0 85.3 13.1 [88.75-99.17] [75.3-92.4] [10.0-17.1] FluAS01E 75 93.3 80.0 14.5 [85.12-97.80] [69.2-88.4] [10.4-20.2] B/Jiangsu (B) Flu Yng 75 100  81.3 13.9  [95.20-100]  [70.7-89.5] [10.1-19.1] Flu Elderly 49 93.9 44.94.3 [83.13-98.72] [30.7-59.8] [3.0-6.1] FluAS01B 75 100   65.3 5.2[95.20-100]  [53.5-76.0] [4.2-6.5] FluAS01E 75 97.3 70.7 6.7[90.70-99.68] [59.0-80.6] [5.1-8.9] N = total number of subject; % =Percentage of subjects with titre at day 21 within the specified range;CI = confidence interval

VI.3.2. Immunogenicity Conclusions

-   -   Pre-vaccination frequency of influenza-specific CD4 was        significantly inferior in elderly adults compared to adults aged        between 18 and 40 years. After vaccination with Fluarix™,        post-vaccination frequency (day 21) remained inferior in elderly        adults compared to younger ones. On the contrary, the        non-inferiority in term of frequency of post-vaccination        frequency of influenza-specific CD4 after vaccination with        adjuvanted vaccines of elderly subjects was demonstrated        compared to vaccination with Fluarix™ in adults aged between 18        and 40 years.    -   Regarding the humoral immune response in term of HI antibody        response, all influenza vaccines fulfilled the requirements of        the European authorities for annual registration of influenza        inactivated vaccines [“Note for Guidance on Harmonisation of        Requirements for Influenza Vaccines for the immunological        assessment of the annual strain changes” (CPMP/BWP/214/96)]. In        elderly adults, adjuvanted vaccines mediated at least a trend        for a higher humoral immune response to influenza haemagglutinin        than Fluarix™ Significant difference between the humoral immune        response against each vaccine strain mediated in elderly        subjects by adjuvanted vaccines compared to Fluarix™ are        summarised in Table 13. Compared to adults aged between 18 and        40 years vaccinated with Fluarix™ elderly subjects vaccinated        with the adjuvanted vaccines showed a trend for higher        post-vaccination GMTs and seroconversion factor at day 21        against the A/New York strain.

TABLE 13 Influenza strains for which significantly higher humoral immunresponse (based on non-overlapping of 95% CI) was observed in elderlysubjects vaccinated with the different adjuvanted vaccines compared toFluarix in the same population. Post-vacc GMT Seroconversion FactorSeroprotection rate Seroconversion Rate FluAS01B A/New York A/NewCaledonia FluAS01E A/New Caledonia A/New Caledonia A/New York Post-vaccGMT = Geometric Mean Titre at post-vaccination

VI.4 Reactogenicity Conclusions

VI.4.1. Recording of Adverse Events (AE)

Solicited symptoms (see Table 14) occurring during a 7-day follow-upperiod (day of vaccination and 6 subsequent days) were recorded.Unsolicited symptoms occurring during a 21-day follow-up period (day ofvaccination and 20+3 subsequent days) were also recorded. Intensity ofthe following AEs was assessed as described in Table 15.

TABLE 14 Solicited local/general adverse events Solicited local AEsSolicited general AEs Pain at the injection site Fatigue Redness at theinjection site Fever Swelling at the injection site Headache HaematomaMuscle ache Shivering Joint pain in the arm of the injection Joint painat other locations

-   N.B. Temperature was recorded in the evening. Should additional    temperature measurements performed at other times of day, the    highest temperature was recorded.

TABLE 15 Intensity scales for solicited symptoms in adults Adverse EventIntensity grade Parameter Pain at injection site 0 Absent 1 on touch 2when limb is moved 3 prevents normal activity Redness at injection siteRecord greatest surface diameter in mm Swelling at injection site Recordgreatest surface diameter in mm Haematoma at injection site Recordgreatest surface diameter in mm Fever* Record temperature in ° C./° F.Headache 0 Absent 1 is easily tolerated 2 interferes with normalactivity 3 prevents normal activity Fatigue 0 Absent 1 is easilytolerated 2 interferes with normal activity 3 prevents normal activityJoint pain at the injection 0 Absent site and other locations 1 iseasily tolerated 2 interferes with normal activity 3 prevents normalactivity Muscle ache 0 Absent 1 is easily tolerated 2 interferes withnormal activity 3 prevents normal activity Shivering 0 Absent 1 iseasily tolerated 2 interferes with normal activity 3 prevents normalactivity *Fever is defined as axillary temperature ≥37.5° C. (99.5° F.)

The maximum intensity of local injection site redness/swelling is scoredas follows:

0 is 0 mm; 1 is >0-≤20 mm; 2 is >20-≤50 mm; 3 is >50 mm.

The maximum intensity of fever is scored as follows:

1 is >37.5-≤38.0° C.; 2 is >38.0-≤39.0° C.; 3 is >39.0

The investigator makes an assessment of intensity for all other AEs,i.e. unsolicited symptoms, including SAEs reported during the study. Theassessment is based on the investigator's clinical judgement. Theintensity of each AE recorded is assigned to one of the followingcategories:

1 (mild)=An AE which is easily tolerated by the subject, causing minimaldiscomfort and not interfering with everyday activities;

2 (moderate)=An AE which is sufficiently discomforting to interfere withnormal everyday activities;

3 (severe)=An AE which prevents normal, everyday activities (Inadults/adolescents, such an AE would, for example, prevent attendance atwork/school and would necessitate the administration of correctivetherapy).

VI.4.2. Recording of Adverse Events (AE)

In elderly subjects, the reactogenicity observed with adjuvantedvaccines, in terms of both local and general symptoms was higher thanwith Fluarix™. Not only the incidence but also the intensity of symptomswas increased after vaccination with adjuvanted vaccines (FIG. 8). Grade3 symptoms showed a trend to be higher in the group who received thevaccine adjuvanted with the highest immunostimulants (MPL, QS21)concentration compared to the group who received the adjuvanted vaccinewherein the immunostimulants is at a lower concentration. In all cases,symptoms however resolved rapidly.

Example VII Pre-Clinical Evaluation of Adjuvanted HPV Vaccines in Mice

This study used a bivalent antigenic composition from humanpapillomavirus (HPV), combining virus like particles (VLPs) formed fromL1 of HPV 16 and L1 from HPV 18 as the antigen. The objective of thestudy was to compare the efficacy of this antigenic preparation whenformulated with AS01B and a 1/5 dilution of AS01B, benchmarked againstthe current adjuvant found in GSK's cervical cancer vaccine, AS04 (MPLon alum).

VII.1—Vaccination

Mice (n=12 per group) were injected at 0 and 28 days with vaccineformulations composed of HPV16/18 L1 (2 μg or 0.5 μg each) derived fromHi-5 80/80 L process and formulated with AS04 (50 μg MPL formulated withalum or AS01B (50 μg QS21-50 μg MPL in 0.5 ml) 1/10 and 1/50 Human dose.As the studies were carried out in mice, 1/10 human dose can be taken tobe equivalent to the AS01B human formulation, i.e. 50 μg QS21 and 50 μgMPL in 0.5 ml and 1/50 can be taken to be a 1/5 dilution of the AS01Bhuman formulation i.e. 10 μg QS21 and 10 μg MPL in 0.5 ml. Blood sampleswere taken at 14 and 45 days post dose 11 to assay for total anti-L1type specific antibodies in individual sera. Intracellular cytokinesstaining were measured at days 7 and 14 post II on PBMC and at day 45post II using spleen cells. Frequency of VLPs specific memory B cellswere measured at day 45 post II using spleen cells.

VII.2—Anti-HPV 16/18 L1 ELISA

Quantification of anti-HPV-16 and HPV-18 L1 antibodies was performed byELISA using HPV-16 and HPV-18 L1 as coating. Antigens were diluted at afinal concentration of 0.5 μg/ml in PBS and were adsorbed overnight at4° C. to the wells of 96-wells microtiter plates (Maxisorp Immuno-plate,Nunc, Denmark). The plates were then incubated for 1 hr at 37° C. withPBS containing 1% Bovine Serum Albumine (saturation buffer). Seradiluted in buffer containing PBS+0.1% Tween20+1% BSA were added to theHPV L1-coated plates and incubated for 1 hr 30 min at 37° C. The plateswere washed four times with PBS 0.1% Tween20 and biotin-conjugatedanti-mouse Ig (Dako, UK) diluted at 1/1000 in saturation buffer wasadded to each well and incubated for 1 hr 30 at 37° C. After a washingstep, streptavidin-horseradish peroxydase (Dako, UK), diluted 1/3000 insaturation buffer was added for an additional 30 min at 37° C. Plateswere washed as indicated above and incubated for 20 min at roomtemperature with a solution of 0.04% o-phenylenediamine (Sigma) 0.03%H₂O₂ in 0.1% Tween20, 0.05M citrate buffer pH 4.5. The reaction wasstopped with 2N H2SO4 and read at 492/620 nm. ELISA titers werecalculated from a reference by SoftMaxPro (using a four parametersequation) and expressed in EU/ml.

VII.3—Intracellular Cytokines Staining (ICS)

Intracellular staining of cytokines of T cells was performed on PBL atdays 7 and 14 post II and on spleen cells at day 45 after the secondimmunisation. PBMCs (1 pool/group) or spleen cells (4 pools of 3 organsper group) were collected from mice. In vitro antigen stimulation ofspleen cells were carried out at a final concentration of 5 10⁶ cells/ml(microplate 96 wells) with VLP 16 or 18, (5 μg/ml)+CD49d CD28 antibodies(1 μg/ml) and then incubated 3H at 37° C. Following the antigenrestimulation step, cells were incubated overnight in presence ofBrefeldin (1 μg/ml) at 37° C. to inhibit cytokine secretion. Cellstaining was performed as follows: cell suspensions were washed,resuspended in 50 μl of PBS 1% FCS containing 2% Fc blocking reagent(1/50; 2.4G2). After 10 min incubation at 4° C., 50 μl of a mixture ofanti-CD4-APC (1/50) and anti-CD8 perCp (1/50) was added and incubated 30min at 4° C. After a washing in PBS 1% FCS, cells were permeabilized byresuspending in 200 μl of Cytofix-Cytoperm (Kit BD) and incubated 20 minat 4° C. Cells were then washed with Perm Wash (Kit BD) and resuspendedwith 50 μl of anti-IFγ APC (1/50)+anti-IL-2 FITC (1/50) diluted inPermWash. After 2H incubation at 4° C., cells were washed with Perm Washand resuspended in PBS 1% FCS+1% paraformaldéhyde. Sample analysis wasperformed by FACS. Live cells were gated (FSC/SSC) and acquisition wasperformed on ˜20,000 events (lymphocytes). The percentages of IFγ+ orIL2+ were calculated on CD4+ and CD8+ gated populations.

VII.4—B Cell Memory

Forty-five days after the second immunisation, mice were sacrificed,spleens cells were separated by a lymphoprep gradient (Cedarlane). Bcells were then resuspended in RPMI 1640 medium (Gibco) containingadditives (sodium pyruvate 1 mM, MEM non-essential amino acids,Pen/Strep, Glutamine and (3-2 mercaptoethanol), 5% foetal calf serum, 50U/ml rhIL-2 (eBioscience) and 3 μg/ml CpG. Cells were cultured five daysat a final concentration of 10⁶ cells/ml, in 5 ml per flat-bottomed 6wells. After an activation step with ethanol, nitrocellulose plates(Multiscreen-IP; Millipore) were coated with 10 μg/ml of VLPs or withGoat anti-mouse Ig (GAM; Sigma) diluted 1/200 in PBS. After a saturationstep with complete medium, 100 μl of 2.10⁶ cells/ml were added to VLPscoated plates and 100 μl of 10⁶ and 5.10⁵ cells/ml were added to GAMplates. After an incubation time of 2 hrs at 37° C., plates were storedovernight at 4° C. Plates were washed four times with PBS 0.1% Tween20and anti-mouse Ig Biot diluted 1/200 in PBS 1% BSA 5% FCS (dilutionbuffer) was distributed to plates and incubated for 2 hrs at 37° c.After a washing step, Extravidin HRP (Sigma) diluted 1/550 in dilutionbuffer was added for an additional 1 hr at 37° C. Plates were washed asabove and incubated for 10 min at room temperature with a solution ofAEC (Sigma). Reaction is stopped by rinsing plates gently under tapwater. When plates are dried, read with KS400.

VII.5—Statistical Analysis

The formulation means were compared using a one-way analysis of variance(ANOVA 1). The analysis was conducted on log 10 transformed data fornormalization purpose. When a significant difference between processmeans was detected (p-value 0.05), pair wise comparisons among meanswere performed at a 0.05 significant level (Student-Newman-Keulsmultiple comparison test).

VII.6—Results

Mice were immunized as in VII.1 above. The following groups were used:

Adju- Group Antigen vant Adjuvant dilution 1 HPV 16-18 L1 2 μg AS04 1/10human dose (equivalent to 50 μg MPL per 0.5 ml HD) 2 HPV 16-18 L1 0.5 μgAS04 1/50 human dose (equivalent to 10 μg MPL per 0.5 ml HD) 3 HPV 16-18L1 2 μg AS04 1/10 human dose (equivalent to 50 μg MPL per 0.5 ml HD) 4HPV 16-18 L1 0.5 μg AS04 1/50 human dose (equivalent to 10 μg MPL per0.5 ml HD) 5 HPV 16-18 L1 2 μg AS01B 1/10 human dose (equivalent to 50μg MPL per 0.5 ml HD) 6 HPV 16-18 L1 0.5 μg AS01B 1/50 human dose(equivalent to 10 μg MPL per 0.5 ml HD) 7 HPV 16-18 L1 2 μg AS01B 1/10human dose (equivalent to 50 μg MPL per 0.5 ml HD) 8 HPV 16-18 L1 0.5 μgAS01B 1/50 human dose (equivalent to 10 μg MPL per 0.5 ml HD)

VII.6.1—Humoral Responses

No significant dose range was observed for the two tested doses ofantigens with either dilution of adjuvant for either anti HPV 16-L1antibody titers or anti HPV 18-L1 antibody titers (FIG. 9)

No significant dose range was observed for the two tested doses of eachadjuvant whatever the dose of antigen for anti HPV 16-L1 antibodytiters.

When looking at anti HPV 18-L1 antibody titers, a slight increase intiter was seen for AS01B (1/10 HD) compared to AS01B (1/50 HD) asmeasured at day 14 post II (2.5 fold dose range, p value=0.0035),however this range was observed only for 2 μg antigen and not for 0.5 μgantigen (p value=0.0867), By day 45 post II, no significant dose rangewas seen for the two tested doses of each adjuvant whatever the dose ofantigen.

VII.6.2—Cellular Responses

Intracellular Cytokine Staining

No dose range effect of antigen was observed for the two tested doses ofantigens whatever the dose of adjuvant for HPV 18-L1. Similarfrequencies of VLP16 specific CD4+ T cells were obtained with the twotested doses of antigens with different doses of adjuvants. (FIG. 10).

A slight dosage effect (2.6 fold, p value=0.0009 for HPV 18-L1, 2 fold,p value=0.0187 for HPV 16-L1) was seen for AS01B (1/10 HD) compared toAS01B (1/50 HD), however this range was observed only for 2 μg antigenand not for 0.5 μg antigen.

Specific B Memory Cells

No dose range effect of antigen was observed for the two tested doses ofantigens whatever the dose of adjuvant for HPV 16 or 18 L1 (FIG. 11)

No dose range effect of adjuvant was observed for the two tested dosesof adjuvants whatever the dose of antigen for HPV 17 or 18 L1.

As can be seen from the above results, a 1/5 dilution of AS01B producesa formulation which has equivalent efficacy in immunogenic compositionsto AS01B itself.

Example VIII Preclinical Evaluation of Adjuvanted S. pneumoniae Vaccinesin Mice

The pneumococcal vaccine used in this study was an 11-valent adjuvantedpneumococcal conjugate vaccines (11 PCV/AS) consisting of a mixture of11 pneumococcal polysaccharide conjugates adjuvanted either with AS01Bor AS01E. The conjugates consist of the S. pneumoniae serotypes 1, 3, 4,5, 6B, 7F, 9V, 14, 18C, 19F and 23F purified polysaccharides, eachindividually conjugated to a carrier protein, either diphtheria toxoid(DT), tetanus toxoid (TT) or protein D from H. influenzae (PD). Thevaccines are presented as a freeze-dried powder to be reconstituted withone of the liquid adjuvants.

11PCV/AS is produced as follows:

-   -   The activation and coupling conditions are specific for each        polysaccharide. These are given in the table below. Sized        polysaccharide (except for PS5, 6B and 23F) was dissolved in        NaCl 2M or in water for injection (WFI). The optimal        polysaccharide concentration was evaluated for all the        serotypes. All serotypes except serotype 18C were conjugated        directly to the carrier protein as detailed below.    -   From a 100 mg/ml stock solution in acetonitrile or        acetonitrile/water 50%/50% solution, CDAP (CDAP/PS ratio 0.75        mg/mg PS) was added to the polysaccharide solution. 1.5 minute        later, 0.2M-0.3M NaOH was added to obtain the specific        activation pH. The activation of the polysaccharide was        performed at this pH during 3 minutes at 25° C. Purified protein        (protein D or DT) (the quantity depends on the initial        PS/carrier protein ratio) was added to the activated        polysaccharide and the coupling reaction was performed at the        specific pH for up to 2 hour (depending upon serotype) under pH        regulation. In order to quench un-reacted cyanate ester groups,        a 2M glycine solution was then added to the mixture. The pH was        adjusted to the quenching pH (pH 9.0). The solution was stirred        for 30 minutes at 25° C. and then overnight at 2-8° C. with        continuous slow stirring.    -   Preparation of 18C:    -   18C was linked to the carrier protein via a linker—Adipic acid        dihydrazide (ADH) Polysaccharide serotype 18C was microfluidized        before conjugation.

Derivatization of Tetanus Toxoid with EDAC

For derivatization of the tetanus toxoid, purified TT was diluted at 25mg/ml in 0.2M NaCl and the ADH spacer was added in order to reach afinal concentration of 0.2M. When the dissolution of the spacer wascomplete, the pH was adjusted to 6.2. EDAC(1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) was then added toreach a final concentration of 0.02M and the mixture was stirred for 1hour under pH regulation. The reaction of condensation was stopped byincreasing pH up to 9.0 for at least 30 minutes at 25° C.

Derivatized TT was then diafiltrated (10 kDa CO membrane) in order toremove residual ADH and EDAC reagent.

TT_(AH) bulk was finally sterile filtered until coupling step and storedat −70° C.

Chemical Coupling of TT_(AH) to PS 18C

Details of the conjugation parameters can be found in Table 1.

2 grams of microfluidized PS were diluted at the defined concentrationin water and adjusted to 2M NaCl by NaCl powder addition.

CDAP solution (100 mg/ml freshly prepared in 50/50 v/v acetonitrile/WFI)was added to reach the appropriate CDAP/PS ratio.

The pH was raised up to the activation pH 9.0 by the addition of 0.3MNaOH and was stabilised at this pH until addition of TT_(AH).

After 3 minutes, derivatized TT_(AH) (20 mg/ml in 0.2 M NaCl) was addedto reach a ratio TT_(AH)/PS of 2; the pH was regulated to the couplingpH 9.0. The solution was left one hour under pH regulation.

For quenching, a 2M glycine solution, was added to the mixturePS/TT_(AH)/CDAP.

The pH was adjusted to the quenching pH (pH 9.0).

The solution was stirred for 30 min at 25° C., and then left overnightat 2-8° C. with continuous slow stirring.

Purification of the Conjugates:

The conjugates were purified by gel filtration using a Sephacryl 500HRgel filtration column equilibrated with 0.15M NaCl (S500HR for 18C) toremove small molecules (including DMAP) and unconjugated PS and protein.Based on the different molecular sizes of the reaction components,PS-PD, PS-TT or PS-DT conjugates are eluted first, followed by free PS,then by free PD or free DT and finally DMAP and other salts (NaCl,glycine).

Fractions containing conjugates are detected by UV_(280 nm). Fractionsare pooled according to their Kd, sterile filtered (0.22 μm) and storedat +2-8° C. The PS/Protein ratios in the conjugate preparations weredetermined.

Specific Activation/Coupling/Quenching Conditions of PS S.pneumoniae-Protein D/TT/DTconiugates

1 3 4 7F Serotype μfluid (μfluid.) μfluid 5 6B μfluid PS 2.5 3.0 2.5 7.15.0 5.0 conc. (mg/ml) PS WFI NaCl 2M WFI WFI NaCl 2M NaCl 2M dissolutionPD 10.0 5.0 10.0 5.0 5.0 10.0 conc. (mg/ml) Initial PS/PD 1.5/1 1/11.5/1 1/1 1.1/1 1.2/1 Ratio (w/w) CDAP conc. 0.50 0.75 0.50 0.79 0.830.75 (mg/mg PS) pH_(a) = pH_(c) = pH_(q) 9.0/9.0/9.0 9.0/9.0/9.09.5/9.5/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.5/9.5/9.5

9V 14 18C 19F Serotype μfluid μfluid μfluid μfluid 23F PS 5.0 5.0 4.59.0 2.38 conc. (mg/ml) PS NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 2Mdissolution Carrier 10.0 10.0 20.0 (TT) 20.0 (DT) 5.0 protein conc.(mg/ml) Initial carrier 1.2/1 1.2/1 2/1 1.5/1 1/1 protein/PS Ratio (w/w)CDAP conc. 0.50 0.75 0.75 1.5 0.79 (mg/mg PS) pH_(a) = pH_(c) =9.5/9.5/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 pH_(q)

The 11 conjugates were then mixed together, and the final antigenicpreparation mixed with the appropriate adjuvant before immunisation.

Groups of 40 female 4-weeks old Balb/c mice were immunized IM at days 0,14 and 28 with 0.1 μg of 11-valent PS conjugates formulated with eitherAS01B or AS01E. Anti-PS IgG antibodies were dosed by ELISA in seracollected at day 42.

As can be seen from FIG. 12, comparable responses were seen between thediluted AS01E formulation compared to the AS01B formulation except forPS 14 where a higher response was seen with AS01B, and PS 19F where ahigher response was seen with AS01E.

Example IX Preclinical Evaluation of Adjuvanted and Non-AdjuvantedCytomegalovirus Immunogenic Compositions

IX.1: Guinea Pigs.

IX.1.1 ELISA Anti-gB

Quantification of anti-gB antibodies was performed by ELISA using gB asa coating antigen. Antigen was diluted at a final concentration of 4μg/ml in PBS and 100 μl was incubated overnight at 4° C. in 96 wellmictrotiter plates. Plastes were then saturated for 1 hour at 37° C.with 200 μl of PBS containing 1% bovine serum albumin. Two-fold serialdilutions of sera were added (100 μl/well) and incubated for 1 hour 30minutes at 37° C. The plates were washed 4 times with PBS 0.1% Tween 20and 100 μl of horseradish peroxidase anti-guinea pig IgG (Dako, UK) wasadded to each well and incubated for 1 hour 30 minutes at 37° C. Plateswere washed 4 times with PBS 0.1% Tween 20 and 1 time with water. Thenthey were incubated for 20 min at 22° C. with 100 μl of a solution ofo-phenylenediamine (Sigma) in 0.1M citrate buffer pH 4.2. This reactionwas stopped with 100 μl of H₂SO₄ 2N and read at 490/620 nm. Elisa titerswere determined by interpolation of OD values from a sample reference bySoftMaxPro. Titers were expressed in EU/ml.

Statistical analyses were performed on days 14 post 2 Elisa data usingUNISTAT. The protocol applied for analysis of variance can be brieflydescribed as follows:

-   -   1) Log transformation of the data    -   2) Shapiro-Wilk test on each population (group) in order to        verify the normality 3) Cochran test in order to verify the        homogeneity of variance between different populations (groups)    -   4) Analysis of variance on selected data (one way)    -   5) Tuckey-HSD test for multiple comparison.

IX.1.2—Neutralization Assay

Prior to the assay, MRC5 cells (10000 cells/200 μl MEM medium) weredistributed in 96 well microplates and incubated for 3 days at 37° C.with CO₂. Two-fold dilutions of inactivated sera (30 min at 56° C.) wererealized and incubated with 100 μl of viral solution (800/ml) for 1 hourat 37° C. After incubation, 100 μl of serium/virus mixture wasinoculated in 96 wells microplates containing MRC5 monoloayer. Theplates were centrifuged at 2000RPM for 1 hour at 35° C. After anovernight incubation at 37° C., the plates were fixed with an acetone80% solution (20 minutes at −20° C.). The acetone solution was discardedand CMV positive cells were detected using a specific monoclonalanti-immediate early antigen for 1 hour at 37° C. The plates were washed3 times with PBS and biotin-conjugated anti-mouse Ig was added to eachwell and incubated for 1 hour at 37° C. After a washing step,streptavidin-horseradish peroxidase was added for an addition 30 minutesat 37° C. Plates were washed 4 times and incubated for 10 minutes with asolution of True-blue. Specific coloured signals were recorded byexamination under microscope. Neutralizing titers were expressed as thereverse of the highest dilution of serium giving 50% reduction of CMVpositive cells as compared to a virus control (CMV plus cells withoutserum).

IX.1.3—Immunization Protocols

4 groups were immunised. Each group contained 8 female Hartley Crl:(ha)Guinea pigs 5-8 weeks old, except for a control group (group 4)containing only 4 subjects. Subjects were immunised IM at 0 and 28 days.Serum samples were collected 28 days after the first immunization and 14days after the second immunization. Elisas were performed as describedabove on serum taken at 28 post I and 14 post II. Neutralisation assayswere performed as described above at 14 post II. Groups were as below:

Group Antigen Adjuvant 1 gB NaCl 2 gB AS01B 3 gB AS01E 4 NaCl NaCl

The antigen was prepared as follows: The vaccine antigen is expressed inChinese Hamster Ovary (CHO) cells as gB**, a truncated chimeracontaining peptide sequences from glycoprotein gD of Herpes Simplexvirus 2 (HSV2) at its N and C-terminus. The gB** is truncated at itsC-terminal domain that contains the membrane anchoring sequence and istherefore secreted into the culture supernatant.

For the first three groups, 15 μg gB** made up in 500 μl of either PBS,AS01B or AS01E (prepared as in example 11.2 above) was injectedintramuscularly. In group 4, PBS alone was administered intramuscularly.

IX.1.4—Results

As can be seen in FIG. 13, Significantly higher anti-gB ELISA titreswere observed for the two adjuvanted groups as compared to the gB plain(8 and 5.5-fold higher for gB and AS01B and gb/AS01E respectively). Postdose II antibody titers were very slightly higher (1.5 fold) in thegB/AS01B group compared to the gB/AS01E group.

Multiple comparison: Tuckey—HSD

Group Cases Mean Plain AS01E AS01B Plain 8 4.7917 ** ** AS01E 8 5.5293** AS01B 8 5.6942 **

Plain<AS01E=AS01B

In respect of neutralising titres (FIG. 14):

-   -   No specific neutralising antibodies were observed in the gB        plain group    -   Specific neutralising antibodies were detected in both        adjuvanted groups    -   Similar levels of neutralising antibodies were observed in both        adjuvanted groups.

IX.2—Mice

IX.2.1—ELISA Anti gB

Quantification of anti-gB antibodies was performed by ELISA using gB asa coating antigen. Antigen was diluted at a final concentration of 1μg/ml in PBS and 100 μl was incubated overnight at 4° C. in 96 wellmictrotiter plates. Plastes were then saturated for 1 hour at 37° C.with 200 μl of PBS containing 1% bovine serum albumin. Two-fold serialdilutions of sera were added (100 μl/well) and incubated for 1 hour 30minutes at 37° C. The plates were washed 4 times with PBS 0.1% Tween 20and 100 μl of streptavidin-horseradish peroxidase was added to each wellfor an additional 30 minutes at 37° C. Plates were washed 4 times withPBS 0.1% Tween 20 and 1 time with water. Then they were incubated for 10min at 22° C. with 100 μl of tetra-methyl-benzidine 75% in 0.1M citratebuffer pH 5.8. This reaction was stopped with 100 μl of H₂SO₄ 0.4N andread at 450/620 nm. Elisa titers were determined by interpolation of ODvalues from a sample reference by SoftMaxPro. Titers were expressed inEU/ml.

Statistical analyses were performed on days 14 post 2 Elisa data usingUNISTAT. The protocol applied for analysis of variance can be brieflydescribed as follows:

-   -   1) Log transformation of the data    -   2) Shapiro-Wilk test on each population (group) in order to        verify the normality 3) Cochran test in order to verify the        homogeneity of variance between different populations (groups)    -   4) Analysis of variance on selected data (one way)    -   5) Tuckey-HSD test for multiple comparison.

IX.2.2—Neutralization Assay

Prior to the assay, MRC5 cells (10000 cells/200 μl MEM medium) weredistributed in 96 well microplates and incubated for 3 days at 37° C.with 5% CO₂. Two-fold dilutions (60 μl) of inactivated sera (30 min at56° C.) were incubated with 60 μl of viral solution (8001PU/ml) for 1hour at 37° C. After incubation, 100 μl of sera-virus mixture wasinoculated in 96 well microplates containing MRC5 cells. The plates werecentrifuged at 2000RPM for 1 hour at 35° C. After an overnightincubation at 37° C., the plates were fixed with an acetone 80% solution(20 minutes at −20° C.). The acetone solution was discarded and CMVpositive cells were detected using a specific monoclonal anti-immediateearly I (IE-I)antigen for 1 hour at 37° C. The plates were washed 3times with PBS and biotin-conjugated anti-mouse Ig was added to eachwell and incubated for 1 hour at 37° C. After a washing step,streptavidin-horseradish peroxidase was added for an addition 30 minutesat 37° C. Plates were washed 4 times and incubated for 10 minutes with asolution of True-blue. Specific coloured signals were recorded byexamination under microscope. Neutralizing titers were expressed as thereverse of the high-test dilution of serium giving 50% reduction of CMVpositive cells as compared to a virus control (CMV plus cells withoutserum).

IX.2.3—Intracellular Cytokine Staining

Intracellular detection of T cells cytokines were performed on PBLs ondays 7 and 21 after the second immunization. PBLs were collected frommice and pooled (1 pool per group). In vitro antigen stimulation oflymphocytes (final concentration of 10*7 cells/ml) were done either witha pool of peptide covering the CMV sequence or with the gB protein.PBLs/antigen mix was incubated 2H at 37° C. Cells were then incubatedovernight in the presence of Brefelding (1 μg/ml) at 37° C. to inhibitcytokine secretion.

Cell staining was performed as follows: Cell suspensions were washed,resuspended in 50 μl of PBS 1& FCS containing 2% Fc blocking reagent.After 10 min incubation at 4° C., 50 μl of a mixture of anti-CD4 PE andanti-CD8 perCp was added and incubated 30 minu at 4° C. After a washingstep in PBS 1% FCS, cell membranes were permeabilised by resuspension in200 μl of Cytofix=Cytoperm (kit Beckton Dickinson) and incubated 20 minat 4° C. Cells were then washed with Perm Wash (kit BD) and resuspendedwith 50 μl of an anti-IFN-gamma APC+anti-IL-2 FITC diluted in PermWash.After 2 hours incubation at 4° C., cells were resuspended in PBS 1%FCS+1% paraformaldehyde.

Sample analysis was performed by FACS. Live cells were gated andacquisition was performed on +/−20000 events. The percentages of IFNg+or IL2+ were calculated on CD4+ and CD8+ gated populations.

IX.2.4—Immunisation Protocols

4 groups were immunised. Each group contained 12 female C57B1/6 mice of4-10 weeks old.

Group Antigen Adjuvant 1 gB PBS 2 gB AS01B 3 gB AS01E 4 NaCl NaCl

The antigen was prepared as follows: The vaccine antigen is expressed inChinese Hamster Ovary (CHO) cells as gB**, a truncated chimeracontaining peptide sequences from glycoprotein gD of Herpes Simplexvirus 2 (HSV2) at its N and C-terminus. The gB** is truncated at itsC-terminal domain that contains the membrane anchoring sequence and istherefore secreted into the culture supernatant.

For each group gB** at a concentration of 1.5 μg/dose was made up in 625μl of PBS or adjuvant AS01B or AS01E (prepared as in example 11.2 abovehaving a concentration of 100 μl of immunostimulants per ml or 50 μl ofimmunostimulants per ml respectively). 50 μl (i.e. 1/10 of a human doseof 0.5 ml) was injected intramuscularly. One control group of mice wasinjected with saline. Injections were performed at days 0 and 28. Serumsamples were collected 14 days after the second injections for ELISA andNeutralisation assays. PBLs were collected 7 days and 21 days postsecond injections for ICS.

IX.2.5—Results

Anti-gB ELISA titers (FIG. 15).

A very weak to undetectable level of anti-gB antibodies was observed inthe unadjuvanted gB group. However, a high antibody response (65 and 66fold higher) was observed in both adjuvant groups, AS01B and AS01Erespectively. There was no statistical significance between the AS01Band AS01E group.

Multiple comparision: Tuckety—HSD

Group Cases Mean Plain AS01E AS01B Plain 12 2.1132 ** ** AS01E 12 3.9317** AS01B 12 3.9375 **

Plain<AS01E=AS01B

Anti-CMV Neutralizing Titers (FIG. 16)

Significantly higher anti-gB neutralising titers were observed for thetwo adjuvanted groups as compared to the gB plain group. No significantdifference in neutralising antibody titres was observed between theAS01B and AS01E formulations.

Cell Mediated Immunity.

Due to the very low level of response observed after restimulation of 7post II samples, no discrimination can be done between groups and noconclusive response for CD4 and CD8 stimulation can be seen (FIG. 17).These low to undetectable responses were probably due to a technicalissue during sample preparations. However, responses could be seen 21days post second injection. The CD4 data (FIG. 18) shows no differenceafter restimulation by gB (5 μg/ml) or peptides (2 μg/ml or 4 μg/ml). Asimilar cytokine profile is seen for AS01E and AS01B. No conclusiveresponse can be seen for CD8 stimulation (FIG. 19)

These experiments show that for another antigenic composition and in twodifferent organisms, an adjuvant having lower levels of immunostimulantsis as immunologically effective as that having higher levels.

X: Preclinical Evaluation of Adjuvanted RTS,S Vaccine

X.1—Formulation

The antigenic composition, RTS, S is produced in Saccharomyces cervisiaeand consists of two proteins, RTS and S, that intracellularly andspontaneously assemble into mixed polymeric particulate structures thatare each estimated to contain, on average, 100 polypeptides. RTS is a 51kDa hybrid polypeptide chain of 424 amino acids consisting of 189aaderived from a sporozoite surfact antigen of the malaria parasite P.falciparum strain NF53 (the CSP antigen, a.a. 207 to 395), fused to theamino terminal end of the hepatitis B virus S protein. S is a 24 kDapolypeptide (226 amino acids long) corresponding to the surfact antigenof hepatitis B virus. The lyophilised antigen pellet containsapproximately 50 μg (when designed to be formulated in 0.5 ml withAS01B) or 25 μg (when designed to be formulated in 0.5 ml with AS01E) ofantigen.

AS01B and AS01E were prepared by mixing the various components (PBS,liposomes, MPL and QS21) in a tank, and stirring under asepticconditions. The product was then sterile filtered before filling intovials or syringes. The liquid adjuvant was stored at +2° C. to +8° C.before being used to reconstitute the lyophilised antigen pellet.

X.2—Mice Experiments

Two experiments in mice were performed aiming at comparing the immuneresponses specific to RTS,S induced by RTS,S/AS01B as compared to RTS,Sformulated with AS01E. In each experiment, C57B1/6 mice (10 mice/group)were immunised intramuscularly three times two weeks apart with 10, 5 or2.5 μg of RTS,S formulated with AS01B or AS01E adjuvants. AS controls,two groups were immunised with either AS01B or AS01E alone. The antibodyresponses specific to HBs and CS were assessed for each mouse by ELISA15 days after the third immunisation. The geometric mean antibody titresand their 95% confidence intervals were calculated for all the micereceiving the same treatment in both experiments. Statistical analysesto evaluate adjuvant effect and antigen dose effects were performed onpooled data from both experiments. The CD4 and CD8 specific T cellresponses were measured by flow cytometry 7 days after the second andthird immunizations on pools of blood cells from 5 mice per group. Thustwo values were generated for each group in each experiment.

Humoral Immune Response

As shown in FIGS. 20 and 21, both AS01B and AS01E adjuvants inducestrong comparable antibody responses against CSP and HBs.

A three-way ANOVA on anti-CSP GMTs showed that there was no significantdifferences between AS01B and AS01E for the 5 or 2.5 μg doses of RTS,S.

For the 10 μg dose, AS01B adjuvant was found to induce higher anti-CStiters than AS01E and the GMT ration “AS01B group/AS01E group” was 1.93(95% CI: 1.33-2.79; p=0.001)

Cell Mediated Specific Immune Response

FIGS. 22 and 23 show the levels of CD4 and CD8 T cells specific for CSPand HBs that express IL-2 and/or IFN gamma.

The CD4 response specific for CSP tends to be higher with AS01B ascompared to AS01E after three immunizations whereas the CD8 T cellresponse with AS01E are equivalent to or better than with AS01B.

The CD4 response specific for HBs tends to be higher with AS01B ascompared to AS01E after three immunizations except for the lower dose ofRTS,S where the levels of CD4 T cells are comparable between the twoadjuvants. The HBs specific CD8 T cell responses induced by RTS,Sformulated with AS01E are equivalent to or better than the responsesinduced by RTS,S formulated with AS01B.

These differences are thought to be within the expected variability ofcellular immunology assays.

Pre-clinical evaluation of the RTS,S/AS01E vaccine in mice revealed anacceptable safety profile, similar to that of RTS,S/AS01B.

XI: Clinical Evaluation of RTS,S/AS01E.

Formulations are prepared as in example X above. Sucrose is used as anexcipient in the lyophillised antigen pellet. As in example X, theliquid adjuvant is used to reconstitute the lyophillised antigen. AS01Ewas prepared as described in example 11.2, and stored at +2 to +8° C.until needed for reconstitution.

A phase II randomized double-blind study of the safety andimmunogenicity of RTS,S adjuvanted with AS01E is currently underway inchildren aged 18 months to 4 years living in Gabon. The vaccinationschedule is a 0, 1, 2—month vaccination schedule. Objectives are asfollows for RTS,S/AS01E when administered as 3 doses intramuscularly ona 0,1,2-month schedule to children aged 18 months to 4 years living in amalaria-endemic area:

Coprimary

-   -   to assess safety until one month post Dose 3.    -   To demonstrate non-inferiority to an oil in water emulsion        adjuvanted RTS,S vaccine in terms of anti-CS antibody response        one month post Dose 3.

Secondary

-   -   to assess reactogenicity until one month post Dose 3    -   to demonstrate non-inferiority to an oil in water emulsion        adjuvanted RTS,S vaccine in terms of anti HBs antibody response        one month post Dose 3    -   to describe seroprotection against hepatitis B up to one month        post Dose 3    -   to describe the anti-CS response up to one month post Dose 3

Tertiary

-   -   Safety between 1 month post Dose 3 until 12 months post Dose 3    -   Humoral immune response to CS antigen at 12 months post Dose 3    -   Humoral immune response to HBs antigen at 12 months post Dose 3

Exploratory

-   -   to evaluate T-cell mediated immune response to CS antigen up to        12 months post dose 3    -   to evaluate B-cell memory immune response to CS antigen up to 12        months post dose 3    -   to describe the anti-CS response up to one month post Dose 3        according to documented HBV immunization status at screening.

180 subjects were enrolled, 90 were given a vaccine adjuvanted with apreviously validated proprietary oil in water emulsion adjuvant (termed“control” in the tables below) and 90 were given a vaccine adjuvantedwith AS01E. Healthy male and female children aged 18 months to 4 yearsof age were screened. Vaccines were administered by the IM route to theleft deltoid.

Incidence and Nature of Symptoms (Solicited and Unsolicited) ReportedDuring the 7-Day (Days 0-6) Post-Vaccination Period Following Each Doseand Overall (Total Vaccinated Cohort)

Any symptom General symptoms Local symptoms 95% CI 95% CI 95% CI Group Nn % LL UL N n % LL UL N n % LL UL Dose 1 Gr 1 90 40 44.4 34.0 55.3 90 2325.6 16.9 35.8 90 20 22.2 14.1 32.2 Gr 2 90 47 52.2 41.4 62.9 90 26 28.919.8 39.4 90 32 35.6 25.7 46.3 Dose 2 Gr 1 88 50 56.8 45.8 67.3 88 3640.9 30.5 51.9 88 35 39.8 29.5 50.8 Gr 2 87 53 60.9 49.9 71.2 87 39 44.834.1 55.9 87 34 39.1 28.8 50.1 Dose 3 Gr 1 83 78 94.0 86.5 98.0 83 3441.0 30.3 52.3 83 76 91.6 83.4 96.5 Gr 2 85 82 96.5 90.0 99.3 85 50 58.847.6 69.4 85 79 92.9 85.3 97.4 Overall/dose Gr 1 261 168 64.4 58.2 70.2261 93 35.6 29.8 41.8 261 131 50.2 44.0 56.4 Gr 2 262 182 69.5 63.5 75.0262 115 43.9 37.8 50.1 262 145 55.3 49.1 61.5 Overall/subject Gr 1 90 8796.7 90.6 99.3 90 60 66.7 55.9 76.3 90 83 92.2 84.6 96.8 Gr 2 90 85 94.487.5 98.2 90 70 77.8 67.8 85.9 90 84 93.3 86.1 97.5 Gr.1 = RTS, S.AS01EGr.2 = control LL = lower limit UL = upper limit For each dose andoverall/subject: N = number of subjects with at least one administereddose n/% = number/percentage of subjects presenting at least one type ofsymptom whatever the study vaccine administered For overall/dose: N =number of administered doses n/% = number/percentage of doses followedby at least one type of symptom whatever the study vaccine administered95% CI = exact 95% confidence interval, LL = Lower Limit, UL = UpperLimit

These data demonstrate that an AS01E adjuvanted RTS,S vaccine gaveacceptable reactogenicity results in a paediatric population whencompared with a control formulation.

Serological responses were measured by evaluating antibody responses toHBs and to CSP repeats (anti R32LR). Serum for antibody determinationwas collected at screening, at day 60 and at day 90 (at secondvaccination and at third vaccination). Antibody levels against CS weremeasured by standard ELISA methodology using plate adsorbed R32LRantigen with a standard reference antibody as a control according toSOPs from the laboratory. Results are reported in EU/mL.

Antibody to hepatitis B surface antigen was measured using acommercially available ELISA immunoassay (AUSAB EIA test kit fromAbbott) or equivalent according to the assay instructions. Results arereported in mIU/mL.

Seropositivity Rates and GMCs for Anti-CS Antibodies (Total VaccinatedCohort)

>=0.5 ELU/ML GMC 95% CI 95% CI Antibody Group Timing N n % LL UL valueLL UL Min Max Anti-CS Gr 1 SCREENING 89 0 0.0 0.0 4.1 0.3 0.3 0.3 <0.5<0.5 PII(D60) 78 78 100 95.4 100 81.9 64.9 103.2 4.6 568.6 PIII(D90) 7575 100 95.2 100 215.6 178.8 259.9 14.3 1922.3 Gr 2 SCREENING 90 1 1.10.0 6.0 0.3 0.2 0.3 <0.5 0.5 PII(D60) 78 78 100 95.4 100 56.9 45.7 70.93.6 2380.9 PIII(D90) 80 80 100 95.5 100 164.8 134.1 202.6 6.3 2093.6Gr.1 = RTS, S.AS01E Gr.2 = Control GMC = geometric mean antibodyconcentration calculated on all subjects N = number of subjects withavailable results n/% = number/percentage of subjects with concentrationwithin the specified range 95% CI = 95% confidence interval; LL = LowerLimit, UL = Upper Limit MIN/MAX = Minimum/Maximum

Seropositivity Rates and GMCs for Anti-HBs Antibodies (Total VaccinatedCohort)

>=10 MIU/ML GMC 95% CI 95% CI Antibody Group Timing N n % LL UL value LLUL Min Max Anti-HBs Gr 1 SCREENING 89 43 48.3 37.6 59.2 40.8 23.3 71.4<10.0 46421.6 PII(D60) 78 77 98.7 93.1 100 8936.4 4684.2 17048.7 <10.01615367 PIII(D90) 75 75 100 95.2 100 24527.7 15316.5 39278.5 21.11694306 Gr 2 SCREENING 90 37 41.1 30.8 52.0 20.0 12.8 31.0 <10.0 30796.4PII(D60) 78 77 98.7 93.1 100 3640.0 1963.1 6749.3 <10.0 1508114PIII(D90) 80 80 100 95.5 100 19485.0 13511.3 28099.9 178.6 1103974 Gr.1= RTS, S.AS01E Gr.2 = Control GMC = geometric mean antibodyconcentration calculated on all subjects N = number of subjects withavailable results n/% = number/percentage of subjects with concentrationwithin the specified range 95% CI = 95% confidence interval; LL = LowerLimit, UL = Upper Limit MIN/MAX = Minimum/Maximum

These data demonstrate that an AS01E adjuvanted RTS,S vaccineformulation gave acceptable humoral immune responses in a paediatricpopulation when compared to a validated control.

Example XII Preclinical Evaluation of Varicella Zoster Virus with AS01BCompared to AS01E

The candidate vaccine is composed of a truncated VZV envelope protein,gE, produced in CHO cells.

For this study C57BL/6 mice (n=48) were primed with one human dose (HD)of Varilrix (˜4 log pfu/dose) administered sub-cutaneously. Five weeksafter priming with Varilrix, mice were divided into to 5 groups of 12mice and injected intra-muscularly (tibias) on days 0 and 28 with 5 μgof gE alone, 5 μg gE+AS01E* (1/10 HD) or 5 μg gE+AS01B (1/10 HD). Thecontrol group of mice (primed only) was injected with saline (0.9%NaCl). Immune responses were evaluated at 14 and/or 30 days followingthe second vaccination. Levels of gE specific total antibodies and thefrequency of cytokine producing (1L2/IFN) CD4 and CD8 T cells wereevaluated.

gE specific antibody responses:

An ELISA was developed to detect and quantify gE-specific antibodies inmice sera, using gE protein as the coating antigen. The ELISA titerswere defined as the reciprocal of the serum dilution, which produced anabsorbance (optical density) measure equal to 50% of the maximalabsorbance value. ELISA titers were calculated by regression analysis.

The data demonstrate that gE AS01E and gE AS01B induced similar levelsof gE specific antibodies (pvalues>0.05). Both formulations inducedsignificantly higher responses compared to the gE antigen alone (10-13fold, pvalues<0.05) at both 14 and 30 days post II (FIG. 26).

14 days post II 30 days post II 95% CI 95% CI GMT GMT Group (EU/ml) LLUL (EU/ml) LL UL gE 12067 5960 24433 3832 911 16115 gE/AS01E 12593495504 166059 50439 38071 66825 gE/AS01B 131728 88112 196934 47589 3615862635 Varilrix 34 11 105 33 10 102

gE Specific CD4 and CD8 Responses

Cytokine production was evaluated in CD4 and CD8 T cells using anintra-cellular cytokine staining technique. Spleen cells were isolatedfrom each group of 12 mice at 30 days post II and pooled into 4 groupsof 3 spleens. Spleen cells (1×10⁶) were incubated for 2 hours in thepresence of gE peptides (63 peptides) spanning the complete gE protein(20 aa peptides/10 aa overlap) and then incubated overnight in thepresence of brefeldin. Subsequently cells were stained with fluorescentmAb specific for cell surface CD4/CD8 and following permeabilizationintracellular cytokines IL-2 and IFNγ.

As shown in FIG. 26 although similar cytokine profiles (IL2/IFNγ) wereinduced with both gE AS01B and gE AS01E formulations, the AS01Bformulation induced a higher magnitude of both CD4 and CD8 cytokineproducing cells (2 fold, p>0.05 for CD4, 3.6 fold, p>0.05 for CD8). Dueto unexpectedly high variability of the T cell responses the power todetect a significant difference between adjuvant doses was very limited(<50%). Importantly both gE formulated with AS01B or AS01E inducedcytokine producing CD4 T cells of a significantly higher magnitude (13.3fold, p<0.05) compared to gE alone. Higher levels of CD8 cells were alsoinduced by gE formulated with AS01B or AS01E (3.8 fold, p>0.05) comparedto gE antigen alone.

Example XIII Preclinical Evaluation of AS01B v AS01E in an InfluenzaFerret Model

Materials and Methods

Female ferrets (Mustela putorius furo) aged 4-6 months were obtainedfrom MISAY Consultancy (Hampshire, UK). Ferrets were primed on day 0with heterosubtypic strain H1N1 A/Stockholm/24/90, (4 Log TCID50/ml),250 μl administered intranasally. On day 21, ferrets were injectedintramuscularly with a full human dose (1000 μl vaccine dose, 15 μg HAper A strain, 17.5 μg B strain) of a combination of H1N1 A/New CaledoniaC/20/99 (15 μg/ml), H3N2 A/Wyoming/3/2003 (15 μg/ml) andB/Jiangsu/10/2003 (17.5 μg/ml). Ferrets were then challenged on day 42by intranasal route with 250 μl of a heterosubtypic strain Wh.A/NY/55/04(4.51 Log TCID50/ml).

Vaccinations on day 21 were either with the plain trivalent formulation(“plain” in the tables below) or with the trivalent formulationadjuvanted with AS01B (“AS01B” in the tables below) or AS01E (“AS01E” inthe tables below). Formulations were prepared as set out in example 3above.

Body Temperature Monitoring:

Individual temperatures were monitored during the challenge period andwere assessed using telemetry implants which recorded each individualanimal temperature every 15 minutes before and after the challenge. Allimplants were checked and refurbished and a new calibration wasperformed by DSI before placement in the intraperitoneal cavity. Allanimals were individually housed in single cage during thesemeasurements.

Temperatures were recorded every 15 minutes 6 days before priming until4 days post-priming, as well as 3 days before challenge until 7 dayspost-challenge.

Hemagglutination Inhibition Test (HI).

Test Procedure

Anti-Hemagglutinin antibody titers to the three influenza virus strainswere determined using the hemagglutination inhibition test (HI). Theprinciple of the HI test is based on the ability of specificanti-Influenza antibodies to inhibit hemagglutination of chicken redblood cells (RBC) by influenza virus hemagglutinin (HA). Sera were firsttreated with a 25% neuraminidase solution (RDE) and wereheat-inactivated to remove non-specific inhibitors. After pretreatment,two-fold dilutions of sera were incubated with 4 hemagglutination unitsof each influenza strain. Chicken red blood cells were then added andthe inhibition of agglutination was scored using tears for reading. Thetiters were expressed as the reciprocal of the highest dilution of serumthat completely inhibited hemagglutination. As the first dilution ofsera was 1:10, an undetectable level was scored as a titer equal to 5.

Statistical Analysis

Statistical analysis was performed on HI titers using UNISTAT. Theprotocol applied for analysis of variance can be briefly described asfollowed:

-   -   Log transformation of data.    -   Shapiro-wilk test on each population (group) in order to verify        the normality of groups distribution.    -   Cochran test in order to verify the homogenicity of variance        between the different populations (groups).    -   One-way analysis of variance performed on groups.    -   Tuckey-HSD Test for multiple comparisons.

Viral Titration in Nasal Washes

All nasal samples were first sterile filtered through Spin X filters(Costar) to remove any bacterial contamination. 50 μl of serial ten-folddilutions of nasal washes were transferred to microtiter platescontaining 50 μl of medium (10 wells/dilution). 100 μl of MDCK cells(2.4×10⁵ cells/ml) were then added to each well and incubated at 35° C.for 6-7 days. After 6-7 days of incubation, the culture medium is gentlyremoved and 100 μl of a 1/20 WST-1 containing medium is added andincubated for another 18 hours. The intensity of the yellow formazan dyeproduced upon reduction of WST-1 by viable cells is proportional to thenumber of viable cells present in the well at the end of the viraltitration assay and is quantified by measuring the absorbance of eachwell at the appropriate wavelength (450 nanometers). The cut-off isdefined as the OD average of uninfected control cells—0.3 OD (0.3 ODcorrespond to +/−3 StDev of OD of uninfected control cells). A positivescore is defined when OD is <cut-off and in contrast a negative score isdefined when OD is >cut-off. Viral shedding titers were determined by“Reed and Muench” and expressed as Log TCID50/ml.

Lymphoproliferation Assay.

PBMC were collected by density gradient centrifugation (20 min at 2500rpm and 4° C.) on Ficoll Cedarlane lympholyte mammal solution. PBMC wereresuspended in 5 ml culture medium (RPMI/Add at 4° C.) and 10% of normalferret serum. Additives were composed by 100 mM sodium pyruvate, nonessential amino acids MEM, Penicillin/streptamycine, glutamine and 1000×concentrated β2-mercaptoethanol. Freshly isolated PBMC were immediatelyused for in vitro proliferation assays. The cells were placed in 96-wellflat bottom tissue culture plates at 2×10⁵ cells/well and cultured withdifferent concentrations of antigen (0.1 to 1 μg HA of whole inactivatedvirus) for 44 to 96 h and then were pulse labeled with 0.5 μCi of[³H]thymidine. Incorporation of radiolabel was estimated 4 to 16 h laterby R-emission spectroscopy.

Results

Viral load in nasal washes after challenge.

Nasal washes were collected 2 days before priming (priming=day 0) 1, 2and 7 days post priming, as well as 4 days before challenge(challenge=day 42) and for a period of 7 days post challenge.

Group −2 0 +1 +2 +7 39 42 43 44 45 47 49 Plain 0.82 1.84 5.35 1.85 0.81.82 5.77 4.44 1.97 0.9 AS01E 0.82 2.11 5.83 1.65 0.8 1.62 4.93 4.15 2.40.85 AS01B 0.81 2.26 5.38 1.91 0.82 1.74 2.25 1.89 1.350 0.9

See results in FIG. 27.

Viral Shedding after Priming

A peak of viral shedding was observed in all ferrets 2 days after thepriming.

7 days post priming, only residual viral load was observed in allgroups.

Viral Shedding after Challenge

The peak of viral shedding was observed 24 hours after challenge.

Viral titration 3 days post-challenge showed high viral titers (noprotection) in ferrets immunized with trivalent split plain. Lowerreduction of viral shedding was observed in ferrets immunised withtrivalent split AS01E than was seen with trivalent split adjuvanted withAS01B.

Temperature Monitoring:

Body temperature was monitored from 6 days pre-priming (priming=day 0)until 4 days post-priming as well as from 3 days pre-challenge until 7days post challenge (challenge=day 42). Measurements were taken every 15minutes and an average calculated by mid-day for each group. Results canbe seen in FIG. 28.

Post Priming

Body temperature monitored before, during and after priming did show anincrease in temperature in all groups.

Post-Challenge

Interpretation of body temperature monitoring is difficult. A slightincrease of body temperature was observed post-challenge in ferretsimmunized with trivalent split plain and trivalent split AS01E, but notwith trivalent split AS01B. The score below was obtained by the numberof ferrets with an increase of body temperature >0.4° C.

Increase in Temperature Post Challenge

Trivalent plain: 5/8 (+0.4, +0.4, +0.5, +0.7, +0.8)

Trivalent AS01B 0/8

Trivalent AS01E 6/8 (+0.4, +0.4, +0.5, +0.5, +0.9, +1.6)

This read out is less robust than other read outs used in ferrets.

Haemagglutination Inhibition Test (HI)

Serum samples were collected 4 days before priming, 17 dayspost-priming, 21 days post-immunization and 13 days post challenge.Results can be seen in FIGS. 29 and 30. For all three vaccine strains,statistically significantly higher HI titers were observed in ferretsimmunised with trivalent split adjuvanted with AS01B or AS01E comparedto trivalent split plain. No difference was observed between the twoadjuvanted groups. Compared to other groups statistically significanthigher cross-reactive HI titers to A/New York H3N2 (challenge strain)were observed after immunisation of ferrets with trivalent splitvaccines adjuvanted with AS01B.

What is claimed is:
 1. A method of vaccination of a human individualcomprising delivery of a human dose of an immunogenic compositioncomprising an antigen in combination with an adjuvant, which adjuvantcomprises the QS21 saponin fraction derived from the bark of QuillajaSaponaria Molina presented in the form of a liposome and alipopolysaccharide, wherein said saponin fraction and saidlipopolysaccharide are both present in said human dose at a level ofabout 25 μg, wherein the antigen is selected from the group consistingof an inactivated influenza split virion, tuberculosis M72 antigen, andmalaria RTS, S antigen, and wherein said adjuvant further comprises asterol, wherein the ratio of saponin:sterol is from 1:1 to 1:100 w/w. 2.The method according to claim 1, wherein the ratio of saponin:sterol isfrom 1:1 to 1:5 w/w.
 3. The method according to claim 1, wherein saidsterol is cholesterol.
 4. The method according to claim 1, wherein thelipopolysaccharide is 3D-MPL and the ratio of QS21:3D-MPL is 1:1 w/w. 5.The method of claim 1, wherein the antigen is tuberculosis M72 antigen.6. The method of claim 1, wherein the antigen is the malaria RTS, Santigen.
 7. A method of vaccination of a human individual comprisingdelivery of a human dose of an immunogenic composition comprising atuberculosis M72 antigen in combination with an adjuvant, which adjuvantcomprises the QS21 saponin fraction derived from the bark of QuillajaSaponaria Molina presented in the form of a liposome and alipopolysaccharide, wherein said saponin fraction and saidlipopolysaccharide are both present in said human dose at a level ofbelow 30 μg.
 8. A method of vaccination of a human individual comprisingdelivery of a human dose of an immunogenic composition comprising amalaria RTS, S antigen in combination with an adjuvant, which adjuvantcomprises the QS21 saponin fraction derived from the bark of QuillajaSaponaria Molina presented in the form of a liposome and alipopolysaccharide, wherein said saponin fraction and saidlipopolysaccharide are both present in said human dose at a level ofbelow 30 μg.